From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Control of protein activity by photoinduced spin polarized charge reorganization.

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

Antepartum vaginal Candida colonization and the risk for obstetrical tears.

OBJECTIVE: Vaginal Candida colonization is very common during pregnancy. An association between Candida colonization and obstetrical tears has not yet been investigated. We investigated whether vaginal Candida colonization during pregnancy is associated with an increased risk for obstetrical tears. STUDY DESIGN: A prospective cohort study was undertaken between the years 2014-2016, comparing pregnancy and delivery characteristics of women with and without Candida colonization during pregnancy. Clinical characteristics of Candida positive women and those with normal vaginal flora were collected. To test the statistical significance of the categorical variables, the chi-square test or Fisher's exact test were used, where appropriate. For continuous variables, the Student's t-test was used. RESULTS: During the study period, 102 women with vaginal Candida during pregnancy (of whom 70% had Candida albicans species) and 102 controls with normal vaginal flora were included in the analysis. No significant differences were observed between Candida positive women and those with normal vaginal flora. Pregnancy and delivery outcomes were comparable and no increased risk of obstetrical tears was found in the Candida group. CONCLUSIONS: Vaginal colonization with Candida species during pregnancy was not found to be associated with an increased risk of obstetrical tears.

Pediatric respiratory hospitalizations in small for gestational age neonates born at term.

OBJECTIVE: This study investigates the risk for long-term respiratory hospitalizations of offspring born small for gestational age (SGA) at term. STUDY DESIGN: A retrospective population-based cohort analysis was performed to examine the risk of long-term respiratory hospitalizations between SGA compared to appropriate for gestational age (AGA) newborns. The analysis included all term singleton deliveries occurring between 1991 and 2014 at a single tertiary medical center. Fetuses with congenital malformations, multiple gestation, cases of perinatal mortality and large for gestational age (LGA) were excluded. A Kaplan-Meier survival curve was used to compare cumulative morbidity incidence up to the age of 18 years, and a Cox hazards regression model was used to control for confounders. RESULTS: During the study period 216,671 deliveries met the inclusion criteria; of them 4.8% (n = 10,450) were diagnosed as SGA neonates. During the follow-up period, the rate of hospitalization due to respiratory morbidity was significantly higher in the SGA group as compared to the AGA group (5.2% vs. 4.7%, OR = 1.13, 95% confidence interval [CI] = 1.03-1.24, p = 0.011). The Kaplan-Meier survival curve demonstrated a significantly higher cumulative incidence of respiratory morbidity in the SGA group (log-rank p = 0.026). In the Cox hazards regression model, controlled for relevant clinical confounders, SGA was found to be an independent risk factor for long-term pediatric respiratory morbidity (adjusted hazard ratio [HR] = 1.1, 95% CI = 1.001-1.19, p = 0.049). CONCLUSION: Being delivered SGA at term is an independent long-term risk factor for pediatric respiratory hospitalization.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Maternal history of recurrent pregnancy loss and long-term risk of thromboembolic events.

BACKGROUND: Recurrent pregnancy loss is a multifactorial disorder with potential underlying maternal hypercoagulability. OBJECTIVE: We aimed to investigate whether a history of recurrent pregnancy loss poses an independent risk for future maternal long-term thromboembolic morbidity. STUDY DESIGN: A population-based study compared the incidence of long-term thromboembolic morbidity in a cohort of women with and without a history of recurrent pregnancy loss. Data were collected from two databases that were cross-linked and merged: a computerized hospitalization database and a computerized obstetrics and gynecology perinatal database. Deliveries occurred between the years 1991-2017. The risk for long-term thromboembolic morbidity was based on the hospital's database and a pre-defined set of ICD codes of thromboembolic related hospitalizations. A Kaplan-Meier survival curve was constructed to compare the cumulative incidence of significant thromboembolic morbidity in the exposed and unexposed groups, and a Cox proportional hazards model, to control for confounders. RESULTS: During the study period, 123,791 parturients met the inclusion criteria; 6.7 % n = 8247 of which experienced previous recurrent pregnancy loss-the exposed group. The rate of thromboembolic related hospitalizations was almost double in exposed women as compared to non-exposed 1.1 % vs. 0.6 %, OR = 1.8; 95 % 1.4-2.2; p < 0.001). Cumulative thromboembolic event incidence, as depicted in the survival curves, was also significantly higher among patients with recurrent pregnancy loss history (Log rank p < 0.001). The Cox model confirmed recurrent pregnancy loss history to be an independent risk factor for later maternal thromboembolic morbidity, while controlling for maternal age, known thrombophilia, ethnicity, smoking, hypertension, diabetes, and fertility treatment (aHR 1.27, 95 %CI 1.02-1.59, p=0.034). CONCLUSION: A history of recurrent pregnancy loss is an independent risk factor for long-term thromboembolic morbidity, even in the absence of known maternal thrombophilia.

Pelvic floor disorders following different delivery modes-a population-based cohort analysis.

INTRODUCTION AND HYPOTHESIS: Identifying protective factors against pelvic floor disorders (PFDs) is important. We investigated whether cesarean delivery (CD) at the indication of abnormal second stage of labor (ASSL) has the same protective effect against future PFDs. METHODS: This population-based cohort study included deliveries occurring from 1991-2017 in a tertiary medical center. Women were grouped by their delivery mode: patients with vaginal deliveries (VD) only; those with CD only, excluding second-stage indications; and those with CD due to ASSL. The outcome measure, PFDs and related repair diagnoses, included any recorded hospitalization involving a pre-defined set of ICD-9 codes. A Kaplan-Meier survival curve compared cumulative PFD morbidity in the different groups, and a Cox proportional hazards model controlled for confounders. RESULTS: A total of 106,003 patients met the inclusion criteria; 86.7% (n = 91,856) experienced VD only, 11.7% (n = 12,359) underwent CD only and 1.7% (n = 1788) had at least one CD due to ASSL. PFD-related hospitalization incidence was 0.7% (n = 719) for the entire cohort. While a significantly higher PFD-related hospitalization rate was recorded in the VD group (0.7%) compared with the ASSL CD group (0.3%) and the non-ASSL CD group (0.5%, p < 0.001), after controlling for potential confounders, an independent and protective association was noted between CD and later PFDs only in parturients who did not experience ASSL (aHR 0.679, 95% CI 0.51-0.90, p = 0.006). CONCLUSIONS: CDs may be protective against later PFD development only if performed prior to the second stage of labor.

Meconium-stained amniotic fluid exposure is associated with a lower incidence of offspring long-term infectious morbidity.

OBJECTIVE: Meconium-stained amniotic fluid (MSAF) is a well-established risk factor for immediate adverse neonatal outcomes and was recently suggested to be associated with microbial invasion of the amniotic cavity. We aimed to determine whether MSAF exposure during labor carries a longer lasting impact on pediatric infectious morbidity. STUDY DESIGN: A population-based cohort analysis was performed including all singleton deliveries occurring between 1991 and 2014 at a single tertiary medical center. Exposure was defined as the presence of MSAF during labor. Hospitalizations of the offspring up to the age of 18 years involving infectious diseases were evaluated. A Kaplan-Meier survival curve was used to compare cumulative morbidity and a Cox regression model to control for confounders. RESULTS: During the study period, 243 725 deliveries met the inclusion criteria. Of them, 35 897 (14.7%) involved MSAF. Rate of infectious-related hospitalizations of the offspring was significantly lower in children exposed to MSAF as compared with the unexposed group (10.8% vs 11.1%, P < 0.05). Specifically, hospitalizations involving respiratory infections were significantly less common among the MSAF group (5.1% vs 5.6%, P < 0.001). The survival curve demonstrated significantly lower cumulative total infectious morbidity rates in the MSAF-exposed group (log rank P < 0.001). In the Cox model, controlled for maternal age, diabetes, hypertension, mode of delivery, and gestational age, exposed children exhibited lower rates of long-term childhood infectious morbidity (adjusted HR 0.96, 95% CI 0.92-0.99, P < 0.001). CONCLUSION: Fetal exposure to MSAF during labor and delivery appears to be associated with lower rates of long-term infectious-related hospitalizations in the offspring.

Maternal recurrent pregnancy loss is associated with an increased risk for long-term neurological morbidity in offspring.

AIM: To determine whether maternal history of recurrent pregnancy loss (RPL) impacts on the long-term neurological health of the offspring. METHOD: A population-based cohort analysis was performed, including all singletons born between 1991 and 2014 at a single regional tertiary centre. Neurological morbidity up to age 18 years (including cognitive, motor, and psychiatric disorders) in children born to mothers with and without a history of RPL was compared. Cumulative neurological morbidity incidence was compared with survival curves and a Weibull multivariable survival model to control for follow-up time and relevant confounders. RESULTS: A total of 242 187 deliveries met the inclusion criteria during the study period, 5% (n=12 182) of which were in mothers with RPL. Epilepsy and developmental disorders were significantly more common in the group with RPL (0.95 vs 0.74/1000 person-years [p=0.009] and 0.22 vs 0.09/1000 person-years [p<0.001]). The survival curves demonstrated significantly higher cumulative incidences of epilepsy and developmental disorders in the group with RPL. The multivariable model exhibited an independent association between maternal RPL and childhood epilepsy (adjusted hazard ratio 1.23; 95% confidence interval 1.01-1.50) and developmental disorders in the offspring (adjusted hazard ratio 2.41; 95% confidence interval 1.60-3.64). INTERPRETATION: A history of maternal RPL appears to be independently associated with long-term neurological morbidity of the offspring. WHAT THIS PAPER ADDS: Hospitalizations for epilepsy and developmental disorders are significantly more common among children of mothers with a history of recurrent pregnancy loss.

Prevalence and risk factors for urinary tract infection following stress urinary incontinence surgery with two midurethral sling procedures.

OBJECTIVE: To compare prevalence and risk factors for urinary tract infection (UTI) following midurethral sling surgery with either the GYNECARE ABBREVO Continence System (Ethicon, Somerville, NJ, USA) or a standard transobturator. METHODS: A retrospective cohort study was conducted among women who underwent midurethral sling surgery to treat stress urinary incontinence at a single tertiary hospital in Israel between January 1, 2014, and August 11, 2015. Data were retrieved from medical records. The diagnosis of UTI was based on a positive urine culture result. RESULTS: Of 178 patients included, 30 (16.9%) underwent the ABBREVO procedure and 148 (83.1%) underwent the standard transobturator procedure. The mean ± SD BMI at the time of surgery was 28.1 ± 4.5 for the standard transobturator procedures and 30.7 ± 15.2 for the ABBREVO procedures. Women who underwent the ABBREVO procedure had an increased duration of hospitalization (P=0.004), and higher rates of concomitant anterior colporrhaphy procedures (P=0.009) and concomitant hysterectomy (P=0.009). Only 38 (21.3%) women developed UTIs within 12 months of surgery (seven in the ABBREVO procedure group and 31 in the standard transobturator procedure group). No statistically significant between-group differences were found for the risk of UTI or for urinary tract adverse events, such as recurrent UTI and the need for rehospitalization. CONCLUSIONS: The two procedures were comparable in terms of prevalence and risk factors for UTI during the postoperative period.

Prevalence and risk factors for urinary tract infection up to one year following midurethral sling incontinence surgery.

OBJECTIVE: To investigate the prevalence and risk factors of urinary tract infection (UTI) one year following sub-midurethral sling (SMUS) incontinence surgery in a university affiliated medical center in southern Israel. METHODS: A retrospective cohort study was conducted to identify and characterize patients who suffered UTI within a year following SMUS surgery. The study population comprised of all patients who underwent a SMUS surgery between the years 2014 and 2015. Demographic and clinical data were retrieved from the patients' medical records, and a comparison between patients with and without a positive urine culture was performed. RESULTS: During the study period, there were 178 SMUS surgeries. Urine culture positive UTI was noted in 21% (38 patients) within the first year following surgery. The mean age and BMI of patients complicated with UTI was 64.8 and 29.1, respectively. The most common pathogen found in urine culture was E. coli that accounted for 55% of all UTIs. When comparing patients with and without UTI, no significant difference was noted in the pre- and intra-operative characteristics. However, duration of hospitalization and readmissions in the first year following surgery, were significantly associated with the risk of UTI (p < 0.026 and p < 0.003, respectively). CONCLUSIONS: Approximately one in every five women undergoing a SMUS operation in our population will suffer from UTI within a year from surgery. A significant association was found between the duration of hospitalization and readmissions in the first postoperative year and suffering from UTI.

Evidence that children born at early term (37-38 6/7 weeks) are at increased risk for diabetes and obesity-related disorders.

BACKGROUND: Prematurity is known to be associated with high rates of endocrine and metabolic complications in the offspring. Offspring born early term (37-38 6/7 weeks' gestation) were also shown to exhibit long-term morbidity resembling that of late preterm, in several health categories. OBJECTIVE: We aimed to determine whether early term delivery impacts on the long-term endocrine and metabolic health of the offspring. STUDY DESIGN: A population-based cohort analysis was performed, including all term singleton deliveries occurring from 1991 through 2013 at a single regional tertiary medical center. Congenital malformations and multiple pregnancies were excluded. Gestational age upon delivery was subdivided into early term deliveries and deliveries occurring at full term and later (≥39 weeks' gestation, comparison group). Endocrine and metabolic morbidity (including diabetes, obesity, hypoglycemia, hyperlipidemia, and hypothyroidism) of the offspring, up to the age of 18 years, was evaluated according to hospitalization files. Kaplan-Meier survival curves were used to compare cumulative morbidity incidence. A Weibull parametric survival model was used to control for time to event, siblings, and other confounders. RESULTS: During the study period 225,260 term deliveries met the inclusion criteria. Of them, 24% (n = 54,073) occurred at early term. Endocrine and metabolic morbidity was significantly more common in the early term group (0.51% vs 0.41%, P = .003). Specifically, overweight and obesity were more common among the early term group (P = .002). Differences were more prominent among children >5 years, who exhibited higher rates of type 1 diabetes mellitus, as well as obesity, when born at early term (P < .05). The survival curves demonstrated higher cumulative incidence of total endocrine and metabolic morbidity in the early term group. Using the Weibull parametric survival model, while controlling for siblings, maternal diabetes, hypertension, labor induction, and Apgar score, early term delivery exhibited an independent association with long-term childhood endocrine and metabolic morbidity of the offspring (adjusted hazard ratio, 1.17; 95% confidence interval, 1.01-1.34) and more so for age >5 years (adjusted hazard ratio, 1.30; 95% confidence interval, 1.08-1.56). CONCLUSION: Deliveries occurring at early term are associated with higher rates of long-term pediatric endocrine and metabolic morbidity of the offspring as compared with deliveries occurring at a later gestational age. This association may be due to absence of full maturity of the hormonal axis in early term neonates or, alternatively, suggest an underlying fetal endocrine dysfunction as the initial mechanism responsible for spontaneous early term delivery.

Inflammatory and vascular placental lesions are associated with neonatal amplitude integrated EEG recording in early premature neonates.

INTRODUCTION: Placental histologic examination can assist in revealing the mechanism leading to preterm birth. Accumulating evidence suggests an association between intrauterine pathological processes, morbidity and mortality of premature infants, and their long term outcome. Neonatal brain activity is increasingly monitored in neonatal intensive care units by amplitude integrated EEG (aEEG) and indices of background activity and sleep cycling patterns were correlated with long term outcome. We hypothesized an association between types of placental lesions and abnormal neonatal aEEG patterns. OBJECTIVE: To determine the association between the placental lesions observed in extreme preterm deliveries, and their neonatal aEEG patterns and survival. PATIENTS AND METHODS: This prospective cohort study included extreme premature infants, who were born ≤ 28 weeks of gestation, their placentas were available for histologic examination, and had a continues aEEG, soon after birth)n = 34). Infants and maternal clinical data were collected. aEEG data was assessed for percentage of depressed daily activity in the first 3 days of life and for sleep cycling. Associations of placental histology with clinical findings and aEEG activity were explored using parametric and non-parametric statistics. RESULTS: Twenty two out of the 34 newborns survived to discharge. Preterm prelabor rupture of membranes (PPROM) or chorioamnionitis were associated with placental lesions consistent with fetal amniotic fluid infection (AFI) or maternal under perfusion (MUP) (P < 0.05). Lesions consistent with fetal response to AFI were associated with absence of SWC pattern during the 1st day of life. Fetal-vascular-thrombo-occlusive lesions of inflammatory type were negatively associated with depressed cerebral activity during the 1st day of life, and with aEEG cycling during the 2nd day of life (P<0.05). Placental lesions associated with MUP were associated with depressed neonatal cerebral activity during the first 3 days of life (P = 0.007). CONCLUSIONS: Depressed neonatal aEEG patterns are associated with placental lesions consistent with maternal under perfusion, and amniotic fluid infection of fetal type, but not with fetal thrombo-oclusive vascular disease of inflammatory type. Our findings highlight the association between the intrauterine mechanisms leading to preterm parturition and subsequent depressed neonatal cerebral function early after birth, which eventually may put premature infants at risk for abnormal neurodevelopmental outcome.

Native tissue repair for central compartment prolapse: a narrative review.

Central descent due to a level 1 defect is a main component in pelvic organ prolapse (POP) reconstructive surgery, whether for symptomatic apical prolapse or for the prolapse repair of other compartments. A recent growth in the rate of native tissue repair procedures for POP, following the US Food and Drug Administration (FDA) warnings regarding the safety and efficacy of synthetic meshes, requires a re-evaluation of these procedures. The safety, efficacy, and determination of the optimal surgical approach should be the center of attention. Functional outcome measures and patient-centered results have lately gained importance and received focus. A comprehensive literature review was performed to evaluate objective and subjective outcomes of apical prolapse native tissue repair, with a special focus on studies reporting impact on patients' functional outcomes, quality of life, and satisfaction. We performed a MEDLINE search for articles in the English language by using the following key words: apical prolapse, sacrospinous ligament fixation, uterosacral ligament suspension, sacral colpopexy, McCall culdoplasty, iliococcygeus vaginal fixation, and functional outcomes. We reviewed references as well. Despite a prominent shortage of studies reporting standardized prospective outcomes for native tissue repair interventions, we noted a high rate of safety and efficacy, with a low complication rate for most procedures and low recurrence or re-treatment rates. The objective and subjective results of different procedures are reviewed. Functional outcomes of native tissue repair procedures have not been studied sufficiently, though existing data present those procedures as favorable and not categorically inferior to sacrocolpopexy. Apical compartment prolapse repair using native tissue is not a compromise. Functional outcomes of native tissue repair procedures are favorable, have a high rate of success, improve women's quality of life (QoL), and result in high rates of patient satisfaction. This subject requires further long-term, standardized prospective studies following the International Continence Society/International Urogynecologists Association guidelines for surgical outcomes report, with the focus on patient-centered functional outcomes.

Fruit-surface flavonoid accumulation in tomato is controlled by a SlMYB12-regulated transcriptional network.

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.