High-Titer Production of the Fungal Anhydrotetracycline, TAN-1612, in Engineered Yeasts.

Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered Saccharomyces cerevisiae (baker's yeast) and Saccharomyces boulardii (probiotic yeast) to produce the nonantibiotic fungal anhydrotetracycline, TAN-1612, in synthetic defined media─necessary for clean purifications─through heterologously expressing TAN-1612 genes mined from the fungus, Aspergillus niger ATCC 1015. This was accomplished via (i) a promoter library-based combinatorial pathway optimization of the biosynthetic TAN-1612 genes coexpressed with a putative TAN-1612 efflux pump, reducing TAN-1612 toxicity in yeasts while simultaneously increasing supernatant titers and (ii) the development of a medium-throughput UV-visible spectrophotometric assay that facilitates TAN-1612 combinatorial library screening. Through this multipronged approach, we optimized TAN-1612 production, yielding an over 450-fold increase compared to previously reported S. cerevisiae yields. TAN-1612 is an important tetracycline analogue precursor, and we thus present the first step toward generating novel tetracycline analogue therapeutics to combat current and emerging antibiotic resistance. We also report the first heterologous production of a fungal polyketide, like TAN-1612, in the probiotic S. boulardii. This highlights that engineered S. boulardii can biosynthesize complex natural products like tetracyclines, setting the stage to equip probiotic yeasts with synthetic therapeutic functionalities to generate living therapeutics or biocontrol agents for clinical and agricultural applications.

Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

Ultrasound-guided intrauterine methotrexate injection of a failed gestation occurring post-endometrial ablation: a case report.

BACKGROUND: Pregnancy following endometrial ablation carries significant risks, including spontaneous abortion, preterm delivery, and abnormal placental adherence. Surgical termination of pregnancy following endometrial ablation has an increased risk for failure and complications. CASE: A 29-year-old woman became pregnant following an endometrial ablation. The pregnancy resulted in a missed abortion; due to prior endometrial ablation, dilatation and curettage was felt to have greater than normal risk. Under ultrasound guidance, methotrexate was injected directly into the gestational sac. The pregnancy resolved with no complications. CONCLUSION: There are significant risks associated with pregnancies occurring post-endometrial ablation and with termination of these pregnancies. Ultrasound-guided local injection of methotrexate appears to be a safe and effective option when termination is required.

Repeated seizures lead to altered skilled behaviour and are associated with more highly efficacious excitatory synapses.

People with epilepsy have a high incidence of interictal behavioural problems that appear to be related to the location of their seizure focus. This study investigated a novel test of the hypotheses that repeated seizures result in behavioural deficits and altered performance during the interictal state, and that those behaviours are related to the presence of more highly efficacious excitatory synapses. We tested these hypotheses by first repeatedly eliciting seizures with electric current through indwelling electrodes in the corpus callosum at the level of the caudal forelimb area of sensorimotor neocortex in the rat. We then assessed learned skilled behaviours that primarily utilize the forelimbs on tasks that are sensitive to the functional integrity of that structure. We observed both behavioural deficits and altered kinematic performance in rats that experienced repeated neocortical seizures relative to an electrode-implanted control group. From a separate set of rats, tissue was prepared for quantification of thickness and excitatory synaptic subtypes from neocortical layer V. We observed significantly increased numbers of perforated synapses that make their connections directly onto the dendritic shaft at 3 weeks following the last seizure. Altered reaching behaviours are likely due to neural reorganization in the neocortex including more efficacious synapses.

Self-concept in adolescents with epilepsy: biological and social correlates.

The purposes of this study were to (1) compare self-esteem in teens with epilepsy to the normative mean, and (2) identify which neurologic/epilepsy and social/familial variables are associated with self-esteem. Thirty-seven adolescents (aged 12-18 years) attending a pediatric neurology clinic completed the Piers-Harris 2 Self-Concept Scale, Family Assessment Measure III, Child Attitude to Illness Scale, and a brief questionnaire about current seizure status (frequency, severity, and number of antiepileptic drugs). Neurology clinic charts were reviewed for seizure types, etiology, age at diagnosis, and number of failed therapies. While Total Piers-Harris t score and most subscales did not differ significantly from the normative mean, teens with epilepsy had higher scores on Behavioral Adjustment (P < 0.04) and Physical Appearance and Attributes (P < 0.03). On univariate analysis, number of current antiepileptic drugs (P < 0.05) and Attitude to Illness and Family Function scores (P < 0.02 for both) were significantly associated with self-esteem. On linear regression analysis, only the Family Function score (P < 0.02) and number of antiepileptic drugs (P < 0.05) were associated with total self-concept. We conclude that self-concept in teens with epilepsy is most strongly associated with Family Function. With the exception of current number of antiepileptic drugs used, epilepsy-specific factors are of minimal importance.

Self-concept, attitude toward illness and family functioning in adolescents with type 1 diabetes.

OBJECTIVES: The primary objective of the present study was to assess self-concept in adolescents with type 1 diabetes, and to determine whether this is associated with attitudes toward having chronic disease, family functioning or severity of diabetes. The secondary objective was to assess the impact of family income, sex, age and age at diagnosis on adolescent self-concept. METHODS: A cross-sectional, self-report survey of 48 adolescents with type 1 diabetes (22 boys and 26 girls; mean +/- SD age at time of study 15.2+/-1.7 years [range 12.2 to 18.0 years]; mean age at diagnosis 9.2+/-3.3 years [range 1.3 to 14.9 years]) was performed using the Piers-Harris Children's Self-Concept (PHCSC) scale, second edition; the Child Attitude Toward Illness Scale; and the Family Assessment Measure scale, version III. Demographic information including net family income and a symptom inventory form to assess disease severity was collected. RESULTS: Adolescents' self-concept measured by the PHCSC scale was significantly positively correlated with a more positive attitude toward chronic illness as measured by the Child Attitude Toward Illness Scale. The PHCSC scale was found to have a significant negative correlation with the Family Assessment Measure scale score, indicating that a better self-concept was correlated with enhanced family functioning. Self-concept was not significantly correlated with disease severity, income of family, sex, age at diagnosis, age at time of study, episodes of diabetic ketoacidosis or episodes of hypoglycemia. CONCLUSIONS: Adolescents with better self-concept had more positive attitudes toward their chronic illness and enhanced family functioning. Although no correlation with diabetes disease severity was seen in the study population, interventions aimed at improving adolescent self-concept may have a positive impact on diabetes treatment by improving attitude toward living with type 1 diabetes.

Impairment of long-term associative memory in aging snails (Lymnaea stagnalis).

Age-dependent impairment in learning and memory functions occurs in many animal species, including humans. Although cell death contributes to age-related cognitive impairment in pathological forms of aging, learning and memory deficiencies develop with age even without substantial cell death. The molecular and cellular basis of this biological aging process is not well understood but seems to involve a decline in the aging brain's capacity for experience-dependent plasticity. To aid in resolving this issue, we used a simple snail appetitive classical conditioning paradigm in which the underlying molecular, cellular, and neural network functions can be directly linked to age-associated learning and memory performance (i.e., the Lymnaea stagnalis feeding system). Our results indicate that age does not affect the acquisition of appetitive memory but that retention and/or consolidation of long-term memory become progressively impaired with advancing age. The latter phenomenon correlates with declining electrophysiological excitability in key neurons controlling the feeding behavior. Together, these results present the Lymnaea feeding system as a powerful paradigm for investigations of cellular and molecular foundations of biological aging in the brain.