FORMATION OF THE ETHER BRIDGE IN THE LOLINE ALKALOID BIOSYNTHETIC PATHWAY

Lolines are specialized metabolites produced by endophytic fungi, such as Neotyphodium and Epichloë species, that are in symbiotic relationships with cool-season grasses. Lolines are vital for the survival of the grasses because their insecticidal and antifeedant properties protect the plant from insect herbivory. Although lolines have various bioactivities, they do not have any concomitant antimammalian activities. Lolines have complex structures that are unique among naturally occurring pyrrolizidine alkaloids. Lolines have four contiguous stereocenters, and they contain an ether bridge connecting C(2) and C(7) of the pyrrolizidine ring. An ether bridge connecting bridgehead C atoms is unusual in natural products and leads to interesting questions about the biosynthesis of lolines in fungal endophytes. Dr. Pan, who was a graduate student in Dr. Schardl Lab at University of Kentucky, isolated a novel metabolite, 1-exo-acetamidopyrrolizidine (AcAP). She observed that AcAP was accumulating in naturally occurring and artificial lolO mutants. I synthesized an authentic sample of (±)-AcAP and compared it spectroscopically with AcAP isolated from a lolO mutant to determine the structure and stereochemistry of the natural product. I was also able to grow crystals of synthetic (±)-AcAP, X-ray analysis of which further supported our structure assignment. There were two possible explanations for the fact that a missing or nonfunctional LolO led to the accumulation of AcAP: that AcAP was the actual substrate of LolO, or that it was a shunt product derived from the real substrate of LolO, 1-exo-aminopyrrolizidine (AP), and that was produced only when LolO was not available to oxidize AP. To distinguish between the two hypotheses, I synthesized 2´,2´,2´,3-[2H4]-AcAP. Dr. Pan used this material to confirm that AcAP was an intermediate in loline alkaloid biosynthesis, not a shunt product. To determine the product of LolO acting on AcAP, Dr. Pan expressed LolO in yeast (Saccharomyces cerevisiae). When Dr. Pan fed AcAP (synthesized by me) to the modified organism, it produced NANL, suggesting that LolO catalyzed two C–H activations of AcAP and the formation of both C–O bonds of the ether bridge in NANL, a highly unusual transformation. Dr. Chang then cloned, expressed, and purified LolO and incubated it with (±)-AcAP, 2-oxoglutarate, and O2. He observed the production of NANL, further confirming the function of LolO. Dr. Chang also observed an intermediate, which we tentatively identified as 2-hydroxy-AcAP. In order to determine whether the initial hydroxylation of AcAP catalyzed by LolO occurred at C(2) or C(7), I prepared (±)-7,7-[2H2]- and (±)-2,2,8-[2H3]-AcAP. When Dr. Pan measured the rate of LolO-catalyzed hydroxylation of these substrates under conditions under which only one C–H activation would occur, she observed a very large kinetic isotope effect when C(2) was deuterated, but not when C(7) was deuterated, establishing that the initial hydroxylation of AcAP occurred at the C(2) position. In order to determine the stereochemical course of C–H bond oxidation by LolO at C(2) and C(7) of AcAP, I synthesized trans- and cis-3-[2H]-Pro and (2S,3R)-3-[2H]- and (2S,3S)-2,3-[2H2]-Asp. Feeding experiments with these substrates carried out by both Dr. Pan (Pro) and me (Asp) showed that at both the C(2) and C(7) positions of AcAP, LolO abstracted the endo H atoms during ether bridge formation. In summary, feeding experiments with deuterated (±)-AcAP derivatives and its amino acid precursors have shown that AcAP is an intermediate in loline biosynthesis. We have shown that LolO catalyzes the four-electron oxidation of AcAP at the endo C(2) position first and then the endo C(7) position to give NANL

Cervical amoebiasis mimicking cervical carcinoma: A rare presentation of a common infection

SummaryCervical amoebiasis is an extremely rare diagnosis with only a small number of published case reports. This disease may present as cervical growth mimicking cervical carcinoma. Owing to the similarity of the clinical presentation of bleeding per vagina and per speculum examination showing growth or ulcers, definitive diagnosis is made on microscopic examination only. We present a rare case of cervical amoebiasis in a 28-year-old, multiparous female who presented with a history of vaginal bleeding. The patient was treated with metronidazole and diloxanide furate, after which she recovered. Awareness of this rare entity is important for clinical suspicion and for the pathologist to identify trophozoites and make a diagnosis, preventing unwarranted investigations. Accurate diagnosis also facilitates quick management of a patient; as this disease is an infective pathology that can easily be treated by antibiotics

Evidence and Experience of Procurement in Health Sector Decentralisation

Improving the efficiency, effectiveness, equity and responsiveness of supply chains and procurement processes for pharmaceuticals, vaccines and other health products, which make up a large share of total health expenditure in low and middle-income countries (LMICs), has important implications for health system performance and population health. Decentralised governance of health services provides greater autonomy in planning, management and decision making from national to sub-national level and has occurred in many LMICs largely as a response to the primary healthcare approach promoted by international agencies. Evidence suggests that procurement is more efficient when centralised because of economies of scale and improved purchasing power whilst other health system functions such as financing and planning/budgeting benefit more from local context-specific implementation. Nepal is embarking on a process of decentralisation after adopting a federal approach to local governance. This helpdesk report looks at other countries to summarise key findings and lessons learnt from decentralised procurement

Primary Tuberculosis of Tonsils: A Case Report

Tuberculosis is one of the major causes of ill health and death worldwide. Isolated tuberculosis of tonsil in the absence of active pulmonary tuberculosis is a very rare clinical entity. A 10-year-male child presented with recurrent episodes of upper respiratory tract infections, with 2-3 occurrences per month for the past 6 years. On general physical examination, bilateral tonsils showed grade III enlargement and congestion. Posterior pharyngeal wall was clear. Examination of the chest was within normal limits. Histopathological examination of bilateral tonsils revealed caseating and noncaseating epithelioid cell granulomas with Langhans giant cells. Ziehl-Neelsen stain for acid fast bacillus was positive. Features were consistent with a diagnosis of tuberculosis of tonsils. Tuberculosis of the oral cavity is uncommon and lesions may be either primary or secondary. Early detection and intervention is essential for cure. Isolated and primary tuberculosis of the tonsils in the absence of pulmonary tuberculosis is a rare entity, which prompted us to report this case

Primary squamous cell carcinoma of the fallopian tube masquerading inflammatory mass

Primary squamous cell carcinoma (SCC) of fallopian tube is exceedingly rare. Till date less than 10 cases have been reported to the best of our knowledge [2-10]. Serous adenocarcinoma is the most common primary malignancy arising from the fallopian tube. Primary SCC of fallopian tube is diagnosed after excluding invasion from the rest of the genital tract and metastasis. We are reporting this rare carcinoma in a 62- year -old female who was taken for laparotomy with pre-operative diagnosis of tubo-ovarian abscess

Midline neck swelling mimicking thyroglossal CYST: Report of an uncommon presentation of cysticercous infestatation

Cysticercosis in humans is caused by an infestation by larvae of Taenia solium. The neck is a rare site of the infestation. Here, we present the case of cysticercosis situated in the neck of a 12-years-old child. The midline neck swelling present in the child since 2 years mimicked thyroglossal cyst, both clinically and radiologically. There were no other systemic complaints. Fine Needle Aspiration Cytology (FNAC) of the swelling was done for reaching the final diagnosis. Hence, in all inflammatory/cystic lesions, particularly in endemic areas, the possibility of cysticercosis should be taken into consideration irrespective of age, location, and the size of the lesion

Pilomatricoma of cheek mimicking malignancy: A diagnostic challenge on Fine needle aspiration cytology

Pilomatricoma is a benign adnexal lesion which is often diagnosed on cytology. Sometimes cytomorphology may pose diagnostic challenge and may mimic malignant neoplasm. We present a case of a slow growing nodular swelling on the right upper cheek in a 9-year-old male child with similar diagnostic difficulty on Fine Needle Aspiration Cytology (FNAC)

Granular cell tumour of clitoris: a case report

Granular cell tumour (GCT) is tumours showing neuroectodermal differentiation. It occurs mostly in the dermis and subcutaneous tissue and occurrence in the muscle layer is rare. The most common site is the head and neck region. It has been known to occur in the vulva, with clitoris being a very rare site. Granular cell tumours are slow growing tumours, which are mostly asymptomatic. We report a case of lump in the clitoris in a 42-year-old woman which was managed with surgical excision. Histopathology confirmed the diagnosis of granular cell tumour. Very few cases of granular cell tumour of clitoris have been reported in literature till date. Although mostly benign, the differential diagnosis of granular cell tumour should be borne in mind by the clinicians in cases of clitoral lump

Coexistant extramedullary hematopoiesisin lymph node and non-hodgkin lymphoma with leukemic spill: A rare case report

Extramedullary hematopoiesis (EMH) commonly occurs in the liver and spleen. Non-hepatosplenic EMH in a lymphnode is rare and with co-existing non-Hodgkin lymphoma is even rarer. Careful screening of cytological slides is a must in order not to miss such rare cases of non-hepatosplenic EMH in lymphnodes. We hereby report a rare case of a 75-year-old male with coexisting lymphnode EMH and NHL, that too with the leukemic spill, which has never been reported before

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