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.

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.

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.

A case-control study addressing the population of epidermal and dermal inflammatory infiltrate including neural milieu in primary prurigo nodularis using S-100 and toluidine blue stain and its therapeutic implications.

BACKGROUND: The pathogenesis of prurigo nodularis (PN) is considered to be multifactorial, with numerous cells and cytokines confabulating to produce an aberrant immune response. METHODS: A cross-sectional observational study was done in cases of untreated primary prurigo nodularis cases with histopathological assessment in 49 cases from lesional and nonlesional skin with assessment of epidermal and dermal changes, dermal infiltrate, S-100 and toluidine blue staining to assess the expression of nerve and mast cells. RESULTS: The most common histological changes seen in lesional skin were hyperkeratosis (98%), irregular hyperplasia (69.4%), hypergranulosis (69.4%), subepidermal clefting (6%), vertical collagen bundles (51.0%), and dermal fibrosis (48.9%). Chronic inflammatory infiltrate was seen in all cases (100%) predominantly of lymphocytes (100%) followed by eosinophils (18.4%), plasma cells (8.2%), and neutrophils (2.0%). There was a marked increase in the expression of S-100 (6.92 ± 3.40 vs. 3.94 ± 2.15, P < 0.001) and toluidine blue (4.99 ± 4.47 vs. 1.22 ± 1.28, P < 0.001) in the lesional skin as compared to the nonlesional skin. CONCLUSION: We can infer that the epidermal and dermal pathology in PN is related to the infiltrate of lymphocytes, mast cells, and neural hyperplasia which perpetuate the pathogenesis by triggering the itch-inflammation cycle. Thus, apart from immunosuppressive agents that target lymphocytes and their cytokines, therapy targeted at mast cells and neural proliferation may be needed to treat prurigo nodularis.

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.

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.

Factors Influencing the Efficiency of Public Hospitals in Saudi Arabia: A Qualitative Study Exploring Stakeholders' Perspectives and Suggestions for Improvement.

Objective: Despite an extensive literature on efficiency, qualitative evidence on the drivers of hospital efficiency is scant. This study examined the factors that influence the efficiencies of health service provision in public hospitals in the Kingdom of Saudi Arabia (KSA) and their potential remedies. Design: We employed a qualitative design involving semi-structured interviews conducted between July and September 2019. Participants were purposively selected and included policymakers and hospital managers drawn from districts, regional and national levels. Data were analyzed in Nvivo 12 based on a thematic approach. Setting: Key informants of Ministry of health in the KSA. Results: Respondents identified a range of different factors across the community, facility and the wider health system that influence inefficiencies in public hospitals in KSA. Ineffective hospital management, lack of strategic planning and goals, weak administrative leadership, and absence of monitoring hospital performance was noted to have a profound impact on hospital efficiency. The conditions of healthcare staff in respect to both skills, authority and psychological factors were considered to influence the efficiency level. Further, lack of appropriate data for decision making due to the absence of an appropriate health informatics system was regarded as a factor of inefficiency. At the community level, respondents described inadequate information on the healthcare needs and expectations of patients and the wider community as significant barriers to the provision of efficient services. To improve hospital efficiencies, respondents recommended that service delivery decisions are informed by data on community health needs; capacity strengthening and effective supervision of hospital staff; and judicious resource allocation. Conclusion: The study demonstrates that inefficiencies in health services remain a critical challenge in public hospitals in KSA. Extensive awareness-raising and training on efficient resource utilization among key health systems stakeholders are imperative to improving hospital performance. More research is needed to strengthen knowledge on hospital efficiency in light of the limited data on the topic in KSA and the wider Gulf region.

Extracranial Schwannomas of the Head and Neck: A Literature Review and Audit of Diagnosed Cases Over a Period of Eight Years.

Schwannoma is a benign, slow growing, usually solitary and encapsulated tumor derived from Schwann cells of the nerve sheath. Schwannomas can be divided into central, or intraosseous, and peripheral lesions. The etiology is unknown, but it is postulated that lesions arise by the proliferation of Schwann cells at one point inside the perineurium. Schwannomas may mimic other diseases of the head and neck, such as infection, tumor or metastasis. Extracranial schwannomas are rare; in this study we review a series of 22 cases of schwannomas originating in the head and neck region over a period of eight years. All tumors were benign and well-encapsulated. Tumor size ranged from 0.5 to 9 cm. The age range of patients studied was 15-74 years with a mean age of 35 years and a male predilection (M:F, 2.6:1)was noted. Four cases of schwannomas occurred in the tongue (18.18%) and lower lip (18.18%), three in the nasal cavity (13.64%), two each (9.09%) in the buccal mucosa, parapharyngeal space (9.09%), and eyebrow (9.09%), and one each in the upper lip (4.55%), lateral canthus of the eye (4.55%), intraorbital region (4.55%), submandibular gland (4.55%), and ear (4.55%). Schwannomas can present in a wide variety of sites within the head and neck. The tumor is benign and tend to be asymptomatic for long periods of time. Histopathology is the gold standard for diagnosis. Our study describes the clinicopathologic features of extracranial head and neck schwannomas, highlights the histopathologic features, and discusses pertinent findings with correlation to the present literature. It is important that both clinicians and pathologists be familiar with the uncommon sites of occurrence and the potential pitfalls associated with the diagnosis and management of these tumors.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded ß-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

A prospective study examining the expression of STAT 1, 3, 6 in prurigo nodularis lesions with its immunopathogenic and therapeutic implications.

BACKGROUND: Prurigo nodularis (PN) is a chronic dermatologic condition manifesting as multiple papulonodular lesions occurring on the background of intense pruritus. PN could be primary or secondary. The exact immune pathogenesis of PN is not yet clear, and multiple pathways are proposed with the JAK-STAT pathway rarely being investigated. AIMS: In this study, our aim was to assess the role of Th cells in PN by comparing the expression of STAT 1, 3, and 6 in involved and normal skin of primary prurigo nodularis. METHODS: A total of 49 clinico-histopathologically proven cases of primary prurigo nodularis were included. Two skin biopsies for each patient from lesional and non-lesional skin were stained with STAT 1, 3, and 6, and the nuclear staining pattern in the epidermis was graded as strong, moderate, weak, or none. RESULTS: Statistically significant expression of STAT 3 and STAT 6 staining was seen in the epidermis of the lesional skin as compared to non-lesional skin. CONCLUSION: Our study showed a marked dominance of STAT 3 and STAT 6 staining in the epidermis which signifies that the keratinocytes play an important role in PN and suggest that Th2, Th17, and Th22 cytokines mediate the tissue response in PN.

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.

Structural characterization of DynU16, a START/Bet v1-like protein involved in dynemicin biosynthesis.

The 1.5 Šresolution crystal structure of DynU16, a protein identified in the dynemicin-biosynthetic gene cluster, is reported. The structure adopts a di-domain helix-grip fold with a uniquely positioned open cavity connecting the domains. The elongated dimensions of the cavity appear to be compatible with the geometry of a linear polyene, suggesting the involvement of DynU16 in the upstream steps of dynemicin biosynthesis.

A study of epithelial-mesenchymal transition immunohistochemical markers in primary oral squamous cell carcinoma.

OBJECTIVE: To analyze markers of epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (SCC) using E-cadherin, epithelial membrane antigen (EMA), vimentin, and smooth muscle actin (SMA), and their association with tumor metastasis and grade. STUDY DESIGN: This was a retrospective study, which included 45 diagnosed cases of primary oral SCC with known lymph node status, taken from the archives of the Department of Pathology along with their clinical profile. Histomorphologic evaluation and immunohistochemical (IHC) analysis for E-cadherin, EMA, vimentin, and SMA were performed. IHC expression of these markers was compared with tumor differentiation as well as lymph node metastasis. RESULTS: We observed that reduced E-cadherin expression and positive expression of SMA were significantly higher in tumors having lymph node metastasis and loss of tumor differentiation, respectively. Reduced EMA expression was associated with the presence of lymph node metastasis but not with the histologic differentiation. Vimentin positivity did not show any correlation with lymph node metastasis or histologic differentiation. CONCLUSIONS: This study emphasizes the importance of EMT in the pathogenesis of oral SCC. The use of biomarkers like E-cadherin, EMA, and SMA might be a valuable tool for predicting patient outcomes and therapy.

Encapsulated papillary variant of medullary carcinoma of thyroid with extensive cystic change: an extremely rare presentation.

Papillary variant of medullary carcinoma of the thyroid is an unusual subtype with many diagnostic challenges. The authors report a case of papillary variant of thyroid medullary carcinoma in a 37-year-old female, who presented with complaints of pain in the thyroid nodule for the latter two months. Contrast enhanced computed tomography (CECT) neck revealed an enlarged and heterogeneously enhancing left lobe of thyroid. This was followed by hemithyroidectomy for suspicion of colloid goitre. Gross examination of the cut surface of the thyroid parenchyma had a sponge like appearance. On histopathology a diagnosis of encapsulated papillary variant of medullary carcinoma thyroid was made with the help of special stains and immunohistochemistry (IHC).

Giant virilising adrenal cortical carcinoma.

Androgen secreting adrenocortical carcinoma (ACC) is a very rare disease with a poor prognosis. Approximately 80% of tumors are functional, most commonly secreting glucocorticoids. We herewith report a case of a huge functional ACC of the right adrenal gland in a 33-year-old female who presented with complaints of hirsutism, amenorrhea and an abdominal lump. On abdominal examination a large lump was palpable in the right hypochondrium reaching up to the umbilicus. Contrast-enhance computed tomography (CECT) revealed a mass in the right suprarenal region. The tumor measured 29 cm × 20 cm × 12 cm and weighed 7.8 kg, the largest reported case of ACC in the world to the best of our knowledge.

Epithelioid inflammatory myofibroblastic sarcoma: the youngest case reported.

Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a rare variant of the inflammatory myofibroblastic tumor. It has an aggressive clinical course and a high rate of recurrence. EIMS primarily affects children and young adults. Hereby, we report this entity in a 4-month-old infant who presented with an abdominal mass. Imaging studies revealed a large hypodense mesentery-based lesion involving the right half and mid-region of the abdomen. The mass with an attached segment of the small bowel was excised in toto. Grossly, a large encapsulated tumor was identified arising from the mesentery of the small bowel. The histological examination showed a tumor consisting of epithelioid to spindle cells loosely arranged in a myxoid background with numerous blood vessels and lymphoplasmacytic inflammatory infiltrate. On immunohistochemistry, the tumor cells showed positivity for ALK1 (nuclear), desmin, SMA, CD68, and focal positivity for CD30. A final diagnosis of EIMS of the small intestine was rendered. To the best of our knowledge, this case is the youngest reported case in literature.