A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Maternal multimorbidity - experiences of women seeking asylum during pregnancy and after childbirth: a qualitative study.

BACKGROUND: Many women seeking asylum during pregnancy and after childbirth have ill-health but detection and assessment of all physical, psychological, and social health needs (maternal multimorbidity) are often difficult as part of routine maternity care. Healthcare providers are key for the early identification and management of vulnerable pregnant women who have additional physical, psychological, and social health needs. We sought to explore the impact of the asylum-seeking process, understanding of wellbeing, expressed health needs (in terms of maternal multimorbidity), and the experiences of maternity care of women seeking asylum during pregnancy and after childbirth in Liverpool, United Kingdom. Enabling factors and barriers to access woman-centred care were also explored. METHODS: Key informant interviews (n = 10) and one focus group discussion (n = 4) were conducted with women attending a non-profit charitable pregnancy support group. Transcribed interviews were coded by topic and then grouped into categories. Thematic framework analysis was undertaken to identify emerging themes. RESULTS: The asylum-seeking process negatively impacted women making them feel anxious and depressed with little control or choice over their future. Women reported feeling stressed regarding poor standard of accommodation, low income, dispersal and the uncertainty of their asylum application outcome. Wellbeing during pregnancy and after childbirth was understood to be multifactorial and women understood that their physical health needs were interlinked and negatively impacted by complex psychological and social factors. Women reported that their expectations of maternity services were often exceeded, but information giving, and the use of language interpreters needed to be improved. Women expressed the need for more psychological and social support throughout pregnancy and after childbirth. CONCLUSIONS: A multidisciplinary team, with links and effective referral pathways to maternal mental health and social services, are necessary for women seeking asylum, to ensure a more integrated, comprehensive assessment of maternal multimorbidity and to provide maternity care in a way that meets all health needs.

Pyridoxal-5'-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis.

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

Comparative evaluation of squash smear and frozen section in the intraoperative diagnosis of central nervous system tumours.

BACKGROUND: The squash smear technique was introduced into intra-operative neurosurgical diagnosis as early as 1930. It is becoming increasingly popular in the diagnosis of central nervous system (CNS) lesions, and is fairly accurate even with a small sample. The current study assesses the accuracy and utility of the squash smear and frozen section (FS) techniques in intraoperative consultations. Correlations with histopathological diagnoses are presented. AIMS: To compare two intra-operative diagnostic techniques-squash smear cytology and FS examination-in the context of central nervous system tumours. MATERIALS AND METHODS: A total of 53 cases of CNS tumours were included in the study, and all were subjected to squash smear examination. FS examinations were conducted for 39 of these samples. The results of the two techniques were compared and correlated with histopathological diagnoses. The observed results were then analysed using SPSS software. RESULTS: The most common primary CNS tumours were gliomas and meningiomas (28.3% each). The sensitivity, specificity, positive predictive value and negative predictive value for squash cytology were 86.67%, 87.5%, 81.25% and 91.3%, and for FS were 91.67%, 93.10%, 91.67%, 93.10%, respectively, with a corresponding comparative P-value of 0.56 (insignificant). Cytological diagnosis showed complete correlation with histopathological diagnosis in 39 cases (73.58%), partial correlation in eight cases (15.1%) and no correlation in six cases (11.32%). FS diagnosis showed complete correlation in 29 cases (74.35%), partial correlation in eight cases (20.5%) and no correlation in two cases (5.1%). CONCLUSION: Squash smear is a rapid, self-sufficient and cost-effective method for the intraoperative diagnosis of CNS tumours. The squash smear and FS techniques are complementary procedures that assist the pathologist in reaching a diagnosis.

Enzymatic Cß-H Functionalization of l-Arg and l-Leu in Nonribosomally Derived Peptidyl Natural Products: A Tale of Two Oxidoreductases.

Muraymycins are peptidyl nucleoside antibiotics that contain two Cß-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-ß-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cß-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cß hydroxylation of the Leu residue to generate (2S,3S)-ß-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cß-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

Cutaneous Histoplasmosis in HIV Seronegative Patients: A Clinicopathological Analysis.

BACKGROUND: Diagnosis of extrapulmonary histoplasmosis in HIV seronegative and immunocompetent patients is often challenging, so a high index of suspicion is required. Cutaneous manifestation of infection shows a wide spectrum of lesions including erythematous plaques; maculopapules; crusted, verrucous, or desquamative papules and nodules; abscesses; and mucocutaneous ulcers among others. Due to the variations in its clinical presentation, histopathology plays a very important role in the detection of spores and the confirmation of diagnosis. OBJECTIVES: The aim of our study was to analyze clinicopathological characteristics of cutaneous manifestations of biopsy-proven histoplasmosis in HIV seronegative individuals. We also examined the utility of Fite stain for the diagnosis of Histoplasma capsulatum on tissue biopsy sections. METHODS: This was a retrospective, observational study on 7 patients who were HIV seronegative and clinically manifested with isolated cutaneous lesions or disseminated disease. Skin biopsy from the lesions was performed on all 7 patients. In addition to H&E staining and special stains for detecting fungus, Fite staining was performed on all of the cases to study its utility in detecting H. capsulatum spores. RESULTS: The skin lesions were widely disseminated in all patients and the most common cutaneous lesions were papules, present in all 7 patients. On review of the H&E-stained slides, the most common pattern was histiocytic lobular panniculitis-like infiltrate observed in 4 cases. Fite stain highlighted the yeast as magenta-colored spores on a blue background in all cases, except for 1 with a granulomatous pattern. CONCLUSION: A primary cutaneous manifestation of H. capsulatum infection in non-HIV-infected individuals is extremely rare. Fite stain could aid in differentiating the spores of H. capsulatum from those of other fungi, Cryptococcus and Candida in particular.

Concurrent Thoracic Spinal Intradural Extramedullary Epidermoid and Neurenteric Cyst in a Spinal Dysraphism Child.

INTRODUCTION: Intraspinal epidermoid cysts are congenital or acquired in origin; whereas intraspinal neurenteric cysts (NECs) are of congenital origin. Their individual association with spinal dysraphism and vertebral segmentation anomalies is very well known. CASE PRESENTATION: We hereby report a case of concurrent intradural extramedullary epidermoid and NEC at adjacent vertebral levels in a spinal dysraphism child, not reported in English Literature till now. CONCLUSION: Multiple spinal lesions related to any/all of the 3 germ layers can coexist at same or adjacent vertebral levels in the same patient and surgical planning shown to be done accordingly.

Adrenocortical Carcinoma in a 6-Month-Old Infant.

Adrenocortical carcinoma (ACC) is a rare malignant tumor arising from the adrenal cortex and it is even rarer in infancy. These tumors are usually hormonally active. We report a case of ACC in a 6-month-old infant who was brought by her mother with complaints of hirsutism and abnormally looking genitalia. Serum 17-alpha-hydroxyprogesterone and serum cortisol levels were raised. Subsequently, imaging studies revealed a large well-circumscribed soft-tissue mass in the right suprarenal region. The tumor was excised, and on histopathology in conjunction with immunohistochemistry, a diagnosis of ACC was given. Only four other cases of ACC have been reported till date in the literature, with an age of 6 months or younger. Hereby, we present a case of ACC in a young infant.

Evidence for Modulation of Oxygen Rebound Rate in Control of Outcome by Iron(II)- and 2-Oxoglutarate-Dependent Oxygenases.

Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases generate iron(IV)-oxo (ferryl) intermediates that can abstract hydrogen from aliphatic carbons (R-H). Hydroxylation proceeds by coupling of the resultant substrate radical (R•) and oxygen of the Fe(III)-OH complex ("oxygen rebound"). Nonhydroxylation outcomes result from different fates of the Fe(III)-OH/R• state; for example, halogenation results from R• coupling to a halogen ligand cis to the hydroxide. We previously suggested that halogenases control substrate-cofactor disposition to disfavor oxygen rebound and permit halogen coupling to prevail. Here, we explored the general implication that, when a ferryl intermediate can ambiguously target two substrate carbons for different outcomes, rebound to the site capable of the alternative outcome should be slower than to the adjacent, solely hydroxylated site. We evaluated this prediction for (i) the halogenase SyrB2, which exclusively hydroxylates C5 of norvaline appended to its carrier protein but can either chlorinate or hydroxylate C4 and (ii) two bifunctional enzymes that normally hydroxylate one carbon before coupling that oxygen to a second carbon (producing an oxacycle) but can, upon encountering deuterium at the first site, hydroxylate the second site instead. In all three cases, substrate hydroxylation incorporates a greater fraction of solvent-derived oxygen at the site that can also undergo the alternative outcome than at the other site, most likely reflecting an increased exchange of the initially O2-derived oxygen ligand in the longer-lived Fe(III)-OH/R• states. Suppression of rebound may thus be generally important for nonhydroxylation outcomes by these enzymes.

Epidermolysis bullosa pruriginosa: A rare entity which responded well to thalidomide.

Epidermolysis bullosa pruriginosa is an unusual clinical variant of dystrophic epidermolysis bullosa characterized by sublamina densa blistering and intense pruritus leading to hypertrophic lichenoid nodules, plaques, milia, and variable presence of albopapuloid lesions. Most cases are sporadic but a few cases have autosomal dominant or recessive inheritance. Treatment has been quite disappointing and failed to produce satisfactory or sustained results. We report a case of 39-years-old male with epidermolysis bullosa pruriginosa and its response to thalidomide.

Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions.

The core of the loline family of insecticidal alkaloids is the bicyclic pyrrolizidine unit with an additional strained ether bridge between carbons 2 and 7. Previously reported genetic and in vivo biochemical analyses showed that the presumptive iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, LolO, is required for installation of the ether bridge upon the pathway intermediate, 1- exo-acetamidopyrrolizidine (AcAP). Here we show that LolO is, in fact, solely responsible for this biosynthetic four-electron oxidation. In sequential 2OG- and O2-consuming steps, LolO removes hydrogens from C2 and C7 of AcAP to form both carbon-oxygen bonds in N-acetylnorloline (NANL), the precursor to all other lolines. When supplied with substoichiometric 2OG, LolO only hydroxylates AcAP. At higher 2OG:AcAP ratios, the enzyme further processes the alcohol to the tricyclic NANL. Characterization of the alcohol intermediate by mass spectrometry and nuclear magnetic resonance spectroscopy shows that it is 2- endo-hydroxy-1- exo-acetamidopyrrolizidine (2- endo-OH-AcAP). Kinetic and spectroscopic analyses of reactions with site-specifically deuteriated AcAP substrates confirm that the C2-H bond is cleaved first and that the responsible intermediate is, as expected, an FeIV-oxo (ferryl) complex. Analyses of the loline products from cultures fed with stereospecifically deuteriated AcAP precursors, proline and aspartic acid, establish that LolO removes the endo hydrogens from C2 and C7 and forms both new C-O bonds with retention of configuration. These findings delineate the pathway to an important class of natural insecticides and lay the foundation for mechanistic dissection of the chemically challenging oxacyclization reaction.

Ciliated Foregut Cyst of Gallbladder: A First in Childhood and Review of Literature.

Ciliated foregut cyst (CFC) is a rare developmental anomaly. We report first case of CFC of gallbladder diagnosed in a 9-year-old child. CFCs are usually located above the diaphragm, commonly in a bronchus or oesophagus. A duplex gallbladder was originally suspected, but histology confirmed the CFC. gallbladder.

Langerhans cell disease of the eyelids masquerading as blepharochalasis.

Langerhans cell disease (LCD) occurring as an isolated tumor of the eyelids has been described infrequently. Moreover, none of the reported cases had associated systemic involvement. We present an unusual case of LCD of the eyelids masquerading as blepharochalasis with central diabetes insipidus (CDI).

ANCA positivity in a patient with infective endocarditis-associated glomerulonephritis: a diagnostic dilemma.

Glomerulonephritis (GN) is an immunological phenomenon in bacterial endocarditis. These may be pauci-immune/vasculitic GN, post-infective GN, and sub-endothelial membranoproliferative glomerulonephritis. Each type of glomerulonephritis usually occurs in isolation. We report a case of infective endocarditis with dual existence of pauci-immune/vasculitic GN and post infective type of GN at the same time.

Analysis of Histomorphologic/Molecular Association and Immune Checkpoint Regulators in Epithelioid Glioblastoma and Pleomorphic Xanthoastrocytoma: Are These Tumors Potential Candidates for Immune Checkpoint Blockade?

Accurate diagnosis of Epithelioid glioblastoma (eGB) and pleomorphic xanthoastrocytoma (PXA) is sometimes challenging owing to overlapping histologic and genetic features. There are limited reports on the immune profile of these tumors. In this study, we assessed 21 PXA [15 PXA Grade 2 (PXAG2); 6 PXA Grade 3 (PXAG3)] and 14 eGB for their histopathological and molecular association. Further, their immune profile was compared with GB, IDH1 wild-type (wt) (n-18). Morphologically, PXAG2 mostly differed from eGB; however, it was occasionally difficult to differentiate PXAG3 from eGB due to their epithelioid pattern and less obvious degenerative features. PXAG2 showed predominantly diffuse, whereas variable positivity for epithelial and glial markers was seen in PXAG3 and eGB. All cases showed retained nuclear ATRX and INI-1 . H3K27M or IDH1 mutation was seen in none. P53 mutation was more common in eGB, followed by PXAG3, and least common in PXAG2. BRAF V600E mutation was observed in 66.67% PXAG2, 33.33% PXAG3, and 50% eGB, with 100% concordance between immunohistochemistry (IHC) and sequencing. Thirty-six percent eGB, 33% PXAG3, and 61% PXAG2 harbored CDKN2A homozygous deletion. EGFR amplification was observed in 14% eGB and 66% of GB, IDH wt. PDL1 and CTLA-4 expression was higher in eGB (71.4% and 57.1%), PXAG3 (66.6% and100%), and PXAG2 (60% & 66.7%) as compared with GB, IDH wt (38.8% and 16.7%). Tumor-infiltrating lymphocytes were also observed in a majority of eGB and PXA (90% to 100%) in contrast to GB, IDH wt (66%). This analysis highlights the homogenous molecular and immune profile of eGB and PXA, suggesting the possibility that histologically and molecularly, these two entities represent 2 ends of a continuous spectrum with PXAG3 lying in between. Higher upregulation of PDL1, CTLA-4, and increased tumor infiltrating lymphocytes in these tumors as compared with GB, IDH wt suggests potential candidature for immunotherapy.

Filariasis, with chyluria and nephrotic range proteinuria.

28 yr female presented with (grade III) chyluria, with nephrotic range proteinuria and Ig M mesangial deposition in immunofluorescence, secondary to filariasis which was confirmed by serology and microfilaria in glomerulus,and successfully treated by Renal Pelvic instillation sclerotherapy with 0.2% povidine and medical treatment (Diethylcarbamazine). She was asymptomatic with follow up period of 19 months.

Cancer Stem Cell Markers - CD133 and CD44 - in Paediatric Solid Tumours: A Study of Immunophenotypic Expression and Correlation with Clinicopathological Parameters.

Paediatric solid tumours account for about 30% of all the paediatric malignancies. They differ from adult tumours in various aspects like incidence, etiopathogenesis, biology, response rate and outcome. Immunohistochemical markers such as CD133, CD44, CD24, CD90, CD34, CD117, CD20 and ALDH 1 (aldehyde dehydrogenase-1) have been proposed to detect cancer stem cells in tumours. CD133 is a marker of tumour initiating cells in many human cancers and therefore, it may be possible to develop future therapies by targeting cancer stem cells via this marker. CD44 is a transmembrane glycoprotein also known as homing cell adhesion molecule. It is a multifunctional cell-adhesion molecule and plays an important role in cell-cell interaction, lymphocyte homing, tumour progression and metastasis. In the present study, we assessed the expression of CD133 and CD44 in paediatric solid tumours and correlated their expression with clinico-pathological parameters in paediatric solid tumours. This study was a cross-sectional observational study conducted in the department of pathology at a tertiary care centre. All the histologically diagnosed paediatric solid tumours for a period of one year and four months were retrieved from the archives. The cases were reviewed and included in the study after obtaining informed consent. Immunohistochemistry using the monoclonal antibodies for CD133 and CD44 was performed in the representative tissue sections of all the cases. Immuno-scores were assessed, and the results were compared using Pearson's chi-square test. The present study included 50 cases of paediatric solid tumours. The majority (34%) of the patients were in the age group of less than 5 years, with male preponderance (M:F = 2.3:1). The tumours included were Wilms tumour, yolk sac tumour, rhabdomyosarcoma, lymphoma, neuroblastoma, hepatoblastoma, gastrointestinal stromal tumour (GIST), medulloblastomas, pilocytic astrocytomas, ependymomas and glioblastoma. On immunohistochemical analysis, high expression of CD133 and CD44 was found. A significant association between the expression of CD133 and various tumour groups was observed (p = 0.004). However, CD44 showed variable expression in different tumour groups. Both CD133 and CD44 identified cancer stem cell in paediatric solid tumours. A further validation is warranted to investigate their potential role in therapy and prognosis.