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Background: People with malignancy of undefined primary origin (MUO) have a poor prognosis and may undergo a protracted diagnostic workup causing patient distress and high cancer related costs. Not having a primary diagnosis limits timely site-specific treatment and access to precision medicine. There is a need to improve the diagnostic process, and healthcare delivery and support for these patients. This trial aims to implement and evaluate an optimal model of care for people presenting with MUO to reduce time to diagnosis, improve patient experiences and reduce healthcare costs., Methods/design: This is a pragmatic stepped-wedge cluster randomised trial comparing a control phase of standard practice with an intervention phase. Patient inclusion criteria are: 1) age 18 years or older, 2) presenting with suspected metastatic malignancy without an obvious primary site on imaging, 3) clinically appropriate to undergo diagnostic work-up and 4) able to provide written or verbal consent. The intervention is a new model of care comprising four key components: standardised diagnostic workup, dedicated cancer care coordinators, virtual multidisciplinary meetings and a website resource for patients, carers and clinicians. The primary endpoint is the time to completion of minimum diagnostic workup. Secondary outcomes are whether the type of tumour is diagnosed, clinical trial participation, referral to palliative care, patient-reported physical, social and mental health, patient-reported understanding and uncertainty. Implementation outcomes include acceptability, feasibility, fidelity and adoption and health care use and costs. Intervention implementation will be supported using clinical leadership, education and reinforcement. Patients who consent to having their data collected will receive the model of care active at the site at the time of recruitment. Patients will complete a patient-reported outcomes questionnaire four months after study enrolment. A health economic analysis will be included. Across 15 hospitals, a total sample size of 240 is planned., Discussion: There is a lack of intervention research for people presenting with MUO. The stepped-wedge design seeks to mitigate the potential challenge of enrolling people with a poor prognosis and high symptom burden in trials. This research will generate important evidence with scalability for future research at trial completion., Trial Registration: ACTRN12622001504707., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval was obtained from Peter MacCallum Cancer Centre Human Research Ethics Committee (number 22/141). All methods will be performed in accordance with relevant guidelines and regulations. Participants will provide informed consent. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: There is a disparity in health outcomes between indigenous and nonindigenous Australians, with higher chronic disease burden and shorter life expectancy in this minority population. Although rates of breast cancer among indigenous women are lower than nonindigenous women, they face a higher breast cancer-associated mortality, which may not entirely be explained by socio-economic disadvantage., Methods: This retrospective cohort study investigated previously described pathologic prognostic factors in indigenous Australians in the Northern Territory., Results: Data analyzed confirmed that indigenous women were more likely to have poorer prognostic disease features, including ER/PR negative and human epidermal growth factor receptor 2 amplified tumors, larger tumors, and higher stage disease., Conclusion: These pathologic features portend to a poor prognosis, raising the possibility these factors contribute to the disparity in health outcomes between indigenous and nonindigenous women with breast cancer, in addition to known socio-economic factors., (© 2023 John Wiley & Sons Australia, Ltd.)

Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers where a primary tissue of origin (TOO) is uncertain. Most patients with CUP have limited treatment options and poor survival outcomes. Immune checkpoint inhibitors (ICIs) can be efficacious in some patients with CUP, but the optimal predictive biomarkers are unknown. We therefore assessed immune and genomic biomarkers as well as predicted TOO in patients with CUP, including a subset treated with ICIs., Methods: Patients with CUP were subject to gene-expression profiling (GEP) and DNA panel sequencing. Immune and stromal-related gene expression was explored by NanoString, including genes associated with immunotherapy response (IR) in other solid malignancies. ICI responsive cancer types were assigned based on Food and Drug Administration-approved indications, and either detection of a latent primary tumor or the TOO was suspected based on genomics informed pathology review. Tumor mutation burden (TMB) and gene mutations were also assessed., Results: A total of 219 patients with CUP were included, 215 assessed for TOO in a previous study, with the majority (163) receiving both RNA and DNA tests. Of GEP profiled cases, 33% (59/175) had a high IR gene-expression score. Of the DNA sequenced cases, 16% (32/203) had high TMB (>10 mutations/Mb), including two with mismatch repair deficiency. Low correlation was observed between TMB and an IR score (R=0.26, p<0.001). Among 110 CUPs with a latent primary or suspected TOO, 47% (52/110) belonged to ICI-responsive cancer types. More than half of the CUPs had at least one feature that may predict ICI response (high IR score, high TMB, ICI-responsive cancer type). Among patients with CUP treated with ICIs, 8/28 (29%) responded (2 complete responses and 6 partial responses). Among non-responders, 9 had stable and 11 had progressive disease. All responders had a high IR score (7/8) and/or high TMB (3/8), while most (5/8) belonged to ICI-responsive cancer types. These features were detected at a lower frequency in non-responders and mostly in patients with stable disease., Conclusions: A significant fraction of CUP tumors had genomic features previously associated with ICI response. High IR score was the most sensitive predictive feature of ICI response, warranting evaluation in a larger patient series., Competing Interests: Competing interests: All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare: RJH is a shareholder in Telix Pharmaceuticals and a Founder and Director of PreMIT. CSK is on the advisory board of AstraZeneca, BMS and Roche. CS is on the advisory board of MSD, Sanofi, Janssen, and GSK. ADeF has received grant funding from AstraZeneca, unrelated to this study; no other relationships or activities that could appear to have influenced the submitted work., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: This study aimed to determine the healthcare experiences, quality of life, and psychosocial needs of patients with cancer of unknown primary (CUP) early after diagnosis; comparing their experiences to patients with advanced cancer of a known primary (non-CUP control patients) and published general population reference data where available., Methods: This study was a cross-sectional, multi-site study comparing CUP patients (n = 139) compared to non-CUP controls (n = 45). Demographic, clinical information and patient-reported outcome questionnaire data were collected at baseline., Results: Differences in healthcare experienced were found between CUP and non-CUP controls with CUP patients reporting higher scores for unmet medical communication/information needs compared with non-CUP control patients (p = 0.013) as well as greater uncertainty in illness (p = 0.042). Whilst no differences were found between CUP and non-CUP controls on the EORTC and PROMIS measures, of those that 'received written information about your cancer…' and asked '…how useful was it?' fewer CUP patients reported finding the information useful 40% vs 61%, and more were likely to not have received written information at all 59% vs 32%; (p = 0.002). Additionally, of those that found information about their cancer online, fewer patients with CUP reported finding it useful 32% vs 48% control patients (p = 0.005)., Conclusions: CUP patients have unmet medical communication/information needs and greater uncertainty in illness but do not differ in health-related quality of life domains compared to patients with advanced cancer of a known primary., (© 2022. The Author(s).)

Introduction: Establishment of strong emergency medical services (EMS) systems plays a pivotal role in reducing morbidity and mortality, especially in low and middle-income countries. We aimed to study the EMS utilization and resources available in the ambulances to deliver prehospital care among patients presenting to the Emergency Medicine Department in a tertiary care hospital in south India., Methods: Data regarding prehospital transport practices such as mode of arrival, utilization of EMS, resources available in the ambulance, presenting complaints, triage category, and demographic details were collected and analyzed. Subgroup analysis for time-sensitive complaints was done. Variables were subjected to univariate and multivariate analysis to find the predictors of ambulance usage., Results: The study included 3935 patients. The most common time-sensitive complaints were trauma (17%) and chest pain (11.5%). The most preferred mode of transport was the personal vehicle (45.6%). 29.8% of patients arrived in the ambulance. 97.7% of ambulances were not Advanced Cardiac Life Support equipped and 87.1% did not have an accompanying health care provider. 64.5% inter-hospital patient transfers were through ambulance, 83.8% transfers were unaccompanied. Among patients with time-sensitive complaints, EMS utilization was inadequate (46.8% in acute coronary syndrome, 34% in trauma, and 56.5% in early acute ischemic stroke)., Conclusion: There was underutilization of the EMS services. Majority of the ambulances were not adequately equipped/staffed to deliver prehospital interventions. Policies at national level are required to encourage EMS utilization by the public and urgent measures are needed to improve services provided by them., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Emergencies, Trauma, and Shock.)

The plant Sesbania mosaic virus [a (+)-ssRNA sobemovirus] VPg protein is intrinsically disordered in solution. For the virus life cycle, the VPg protein is essential for replication and for polyprotein processing that is carried out by a virus-encoded protease. The nuclear magnetic resonance (NMR)-derived tertiary structure of the protease-bound VPg shows it to have a novel tertiary structure with an α-β-β-β topology. The quaternary structure of the high-affinity protease-VPg complex (≈27 kDa) has been determined using HADDOCK protocols with NMR (residual dipolar coupling, dihedral angle, and nuclear Overhauser enhancement) restraints and mutagenesis data as inputs. The geometry of the complex is in excellent agreement with long-range orientational restraints such as residual dipolar couplings and ring-current shifts. A "vein" of aromatic residues on the protease surface is pivotal for the folding of VPg via intermolecular edge-to-face π···π stacking between Trp 271 and Trp 368 of the protease and VPg, respectively, and for the CH···π interactions between Leu 361 of VPg and Trp 271 of the protease. The structure of the protease-VPg complex provides a molecular framework for predicting sites of important posttranslational modifications such as RNA linkage and phosphorylation and a better understanding of the coupled folding upon binding of intrinsically disordered proteins. The structural data presented here augment the limited structural data available on viral proteins, given their propensity for structural disorder.

Cancer has become a prevalent disease, affecting millions of new patients globally each year. The COVID-19 pandemic is having far-reaching impacts around the world, causing substantial disruptions to health and health care systems that are likely to last for a prolonged period. Early data have suggested that having cancer is a significant risk factor for mortality from severe COVID-19. A diverse group of medical oncologists met to formulate detailed practical advice on systemic anticancer treatments during this crisis. In the context of broad principles, issues including risks of treatment, principles of prioritizing resources, treatment of elderly patients, and psychosocial impact are discussed. Detailed treatment advice and options are given at a tumor stream level. We must maintain care for patients with cancer as best we can and recognize that COVID-19 poses a significant competing risk for death that changes conventional treatment paradigms.

Hyperplasia of the thymus is commonly seen in myasthenia gravis and other autoimmune disorders. Thymic size also varies with age, corticosteroid use, infections, and inflammatory disease. Although thymic hyperplasia has been described following chemotherapy, there is no known association of true thymic hyperplasia with immune checkpoint inhibitor therapy. We present two cases of suspected true thymic hyperplasia in patients with stage IV melanoma who were treated with the combination of nivolumab and ipilimumab, which was complicated by immune-related toxicity requiring corticosteroid therapy, and then subsequently also by secondary hypoadrenalism requiring replacement hydrocortisone. In one patient, histological and flurocytometric analyses of an incisional biopsy of the thymus revealed findings consistent with true thymic hyperplasia. In the other case, the stable fluorodeoxyglucose positron emission tomography/Computed tomography (FDG-PET/CT) findings were consistent also with true thymic hyperplasia. These are the first described cases of true thymic hyperplasia following combination immune checkpoint inhibitor therapy for metastatic melanoma. We hypothesize that the true thymic hyperplasia in these cases results from initial lymphocyte depletion caused by intense corticosteroid therapy followed by rebound thymic hyperplasia during the period of relative hypocortisolism, which may have been aggravated by the onset of secondary hypoadrenalism., (© 2019 John Wiley & Sons Australia, Ltd.)

Conformational factors that predicate selectivity for valine or isoleucine binding to IlvN leading to the regulation of aceto hydroxy acid synthase I (AHAS I) of Escherichia coli have been determined for the first time from high-resolution (1.9-2.43 Å) crystal structures of IlvN·Val and IlvN·Ile complexes. The valine and isoleucine ligand binding pockets are located at the dimer interface. In the IlvN·Ile complex, among residues in the binding pocket, the side chain of Cys 43 is 2-fold disordered (χ 1 angles of gauche - and trans). Only one conformation can be observed for the identical residue in the IlvN·Val complexes. In a reversal, the side chain of His 53 , located at the surface of the protein, exhibits two conformations in the IlvN·Val complex. The concerted conformational switch in the side chains of Cys 43 and His 53 may play an important role in the regulation of the AHAS I holoenzyme activity. A significant result is the establishment of the subunit composition in the AHAS I holoenzyme by analytical ultracentrifugation. Solution nuclear magnetic resonance and analytical ultracentrifugation experiments have also provided important insights into the hydrodynamic properties of IlvN in the ligand-free and -bound states. The structural and biophysical data unequivocally establish the molecular basis for differential binding of the ligands to IlvN and a rationale for the resistance of IlvM to feedback inhibition by the branched-chain amino acids.

Radiation recall is an uncommon phenomenon describing an acute localised inflammatory toxicity affecting tissue previously exposed to radiotherapy. It is precipitated by administration of certain medications, including chemotherapy. We describe a case involving a 50-year-old Aboriginal male smoker from a remote community in Northern Australia who underwent treatment for stage IV non-small cell lung cancer with localised radiotherapy to the primary right upper lung lobe tumour. This was followed by a course of gemcitabine, which was ceased prematurely after four cycles when he presented with radiation recall to his right pectoralis major. Our case description is complemented with a brief review of current literature regarding our case and gemcitabine-related radiation recall. This was in the context of concurrent musculoskeletal strain, an as-yet unreported association with radiation recall. His condition settled with steroid administration and discontinuation of gemcitabine., (© 2018 The Authors. Journal of Medical Radiation Sciences published by John Wiley & Sons Australia, Ltd on behalf of Australian Society of Medical Imaging and Radiation Therapy and New Zealand Institute of Medical Radiation Technology.)

In the title compound, C21H17N3O3S, the non-H atoms, apart from those in the benzoyl group, are almost coplanar (r.m.s. deviation = 0.049 Å) and the benzoyl group is almost orthogonal to the plane of the rest of the mol-ecule [dihedral angle = 80.34 (6)°]. In the crystal, a combination of N-H⋯O and asymmetric bifurcated O-H⋯(N,O) hydrogen bonds link the mol-ecules into a three-dimensional network. Weak C-H⋯O inter-actions are also observed.

Syntheses and structures are described for some alkylidene-substituted dihydrooxazolones and dihydroimidazoles derived from simple acylglycines. A second, triclinic, polymorph of 4-benzylidene-2-(4-methylphenyl)-1,3-oxazol-5(4H)-one, C17H13NO2, (I), has been identified and the structure of 2-methyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one, C9H7NO2S, (II), has been rerefined taking into account the orientational disorder of the thienyl group in each of the two independent molecules. The reactions of phenylhydrazine with 2-phenyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one or 2-(4-methylphenyl)-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one yield, respectively, 3-anilino-2-phenyl-5-[(thiophen-2-yl)methylidene]-3,5-dihydro-4H-imidazol-4-one, C10H15N3OS, (III), and 3-anilino-2-(4-methylphenyl)-5-[(thiophen-2-yl)methylidene]-3,5-dihydro-4H-imidazol-4-one, C21H17N3OS, (IV), which both exhibit orientational disorder in their thienyl groups. The reactions of 2-phenyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one with hydrazine hydrate or with water yield, respectively, N-[3-hydrazinyl-3-oxo-1-(thiophen-2-yl)prop-1-en-2-yl]benzamide and 2-(benzoylamino)-3-(thiophen-2-yl)prop-2-enoic acid, which in turn react, respectively, with thiophene-2-carbaldehyde to form 2-phenyl-5-[(thiophen-2-yl)methylidene]-3-{[(E)-(thiophen-2-yl)methylidene]amino}-3,5-dihydro-4H-imidazol-4-one, C19H13N3OS2, (V), which exhibits orientational disorder in only one of its thienyl groups, and with methanol to give methyl (2Z)-2-(benzoylamino)-3-(thiophen-2-yl)prop-2-enoate, C15H13NO3S, (VI). There are no direction-specific intermolecular interactions in the crystal structure of the triclinic polymorph of (I), but the molecules of (II) are linked by two independent C-H···O hydrogen bonds to form C2(2)(14) chains. Compounds (III) and (IV) both form centrosymmetric R2(2)(10) dimers built from N-H···O hydrogen bonds, while compound (V) forms a centrosymmetric R2(2)(10) dimer built from C-H···O hydrogen bonds. In the structure of compound (VI), a combination of N-H···O and C-H···π(arene) hydrogen bonds links the molecules into sheets. Comparisons are made with some similar compounds.

Background: Recent data show a falling cancer mortality in the general population without a similar shift in immigrant outcomes, leading to a greater cancer burden and mortality for immigrants. Our aims were to compare perceived patterns of care in immigrants and native-born cancer patients., Patients and Methods: This was a hospital-based sample of first-generation immigrants and Australian-born Anglo patients in the first year following diagnosis. It was restricted to Chinese, Arabic, or Greek speakers. Eligible participants, recruited via 16 oncology clinics, were over 18, with cancer (any type or stage), and having commenced treatment at least 1 month previously. Five hundred and seventy-one CALD patients (comprising 145 Arabic, 248 Chinese, and 178 Greek) and a control group of 274 Anglo-Australian patients participated., Results: Immigrants had difficulty communicating with the doctor (73% versus 29%) and understanding the health system (38% versus 10%). Differences were found in 'difficulty knowing who to see' (P = 0.0002), 'length of time to confirm diagnosis' (P = 0.04), wanting more choice about a specialist and hospital (P < 0.0001); being offered the opportunity to see a counselor (P < 0.0001); and actually seeing one (P < 0.0001). There were no significant self-reported differences regarding how cancer was detected, time to see a health professional, or type first seen; however, immigrants reported difficulty knowing who to see. Previous studies showed differences in patterns of care according to socioeconomic status (SES) and educational level. Despite adjusting for age, sex, education, marital status, SES, time since diagnosis, and type of cancer, we did not find significant differences. Instead, we found that understanding of the health system and confidence understanding English were important factors., Conclusions: This study confirmed that immigrants with cancer perceive an inferior quality of cancer care. We highlight potentially modifiable factors including assistance in navigating the health system, translated information, and cultural competency training for health professionals., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The solution structure of IlvN, the regulatory subunit of Escherichia coli acetohydroxyacid synthase I, in the valine-bound form has been determined using high-resolution multidimensional, multinuclear nuclear magnetic resonance (NMR) methods. IlvN in the presence or absence of the effector molecule is present as a 22.5 kDa dimeric molecule. The ensemble of 20 low-energy structures shows a backbone root-mean-square deviation of 0.73 ± 0.13 Å and a root-mean-square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, more than 98% of the backbone φ and ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map, which is indicative of the fact that the structures are of high stereochemical quality. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain seen in metabolic enzymes. In the valine-bound form, IlvN exists apparently as a single conformer. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR time scale. Thus, a large shift in the conformational equilibrium is observed upon going from the free form to the bound form. The structure of the valine-bound form of IlvN was found to be similar to that of the ACT domain of the unliganded form of IlvH. Comparisons of the structures of the unliganded forms of these proteins suggest significant differences. The structural and conformational properties of IlvN determined here have allowed a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis.

Background: The mainstay of therapy for gastrocutaneous (GC) fistulas has been surgical intervention. However, endoclips are currently used for management of perforations and fistulas but are limited by their ability to entrap and hold the tissue., Objective: To report the first North American experience with a commercially available over-the-scope clip (OTSC) device, a novel and new tool for the endoscopic entrapment of tissue for the closure of fistula and perforations., Methods: The present single-centre study was conducted at a tertiary referral academic gastroenterology unit and centre for advanced therapeutic endoscopy and involved patients referred for endoscopic treatment for the closure of a GC fistula. The OTSC device was mounted on the tip of the endoscope and passed into the stomach to the level of the fistula. The targeted site of the fistula was grasped with the tissue anchoring tripod and pulled into the cap with concomitant scope channel suction. Once the tissue was trapped in the cap, a 'bear claw' clip was deployed., Results: The patients recovered with fistula closure. No complication or recurrence was noted. Fistula sizes >1 cm, however, were difficult to close with the OTSC system. The length of stay of the bear claw clip at the fistula site is unpredictable, which may lead to incomplete closure of the fistula., Conclusion: Closure of a GC fistula using a novel 'bear claw' clip system is feasible and safe.

Production of biosurfactant can be substantially increased by the addition of precursors like vegetable oils, petroleum products, and other water-insoluble substances. Pseudomonas Ptm+ strain produces biosurfactant in the presence of hexachlorocyclohexane (HCH), which specifically emulsifies HCH, a recalcitrant organochlorine pesticide. Addition of previously produced crude biosurfactant by the same organism as a precursor instead of HCH increased production of biosurfactants with a decrease in the total fermentation time from 32 to 24 h. The main objective of this paper was to find alternatives for HCH as an inducer.

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.

Mediastinal lymphadenopathy in a patient with previously treated T-cell acute lymphoblastic leukaemia is a diagnostic problem. The differential diagnosis in an adult is sarcoidosis, metastases, lymphoma or, rarely, tuberculosis. Mediastinal lymph node involvement is uncommon in tuberculosis. In view of its relative rarity but good prognosis, it is important to distinguish tuberculous mediastinal lymphadenitis in adults from other causes of mediastinal masses.

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL(-1). With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible. Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30+/-2 degrees C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis.

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.

Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes 1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70 degrees C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70 degrees C was carried out by using 4% silane and it was found that the enzyme was stable up to 75 degrees C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.

Stability of glucose oxidase (GOD) immobilized with lysozyme has been considerably enhanced by modification of free thiols generated by reducing disulfide bonds using beta-mercaptoethanol and N-ethylmaleimide in conjunction with additives like antibiotics and salts. Thermal stability of immobilized GOD was quantified by means of the transition temperature, Tm and the operational stability by half-life t1/2 at 70 degrees C. Modification of the free thiols in the enzyme coupled with the presence of kanamycin, NaCl, and K2SO4, led to increase in Tm, to 80, 82 and 84 degrees C (compared to 75 degrees C in control) and t1/2 by 7.7-, 11- and 22-fold, respectively, indicating that this method can be effectively used for enhancing the stability of enzymes.

An enzyme inhibitor, nigerloxin, with inhibition against soy bean lipoxygenase-I (LOX-1), rat lens aldose reductase (RLAR) as well as free radical scavenging activity was isolated from the fermented wheat bran using Aspergillus niger CFR-W-105. Its chemical structure was identified as 2-amido-3-hydroxy-6-methoxy-5-methyl-4-(prop-1'-enyl) benzoic acid by NMR and GCEIMS data. The IC50 values against LOX-1 and RLAR were found to be 79 microM and 69 microM and ED50 against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was 66 microM.

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25 +/- 1 degrees C. The soluble enzyme required a GdmCl concentration of 5 M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

Lipase catalysed stearoyl lactic acid preparation in non-aqueous media was treated as a model system to study the microaqueous phase containing hydrogen ions arising from dissociation of water soluble lactic acid in it. The thermodynamic factors operating at the microaqueous enzyme-water-solvent phase on the lipase in non-polar solvents were investigated in terms of the water of reaction which constitutes the microaqueous phase, partitioning of acid between water of the microaqueous phase and the organic solvent, dissolution and dissociation of the acid and the resultant number of H+ present in the microaqueous phase and the extent of esterification for a given amount of enzyme at various substrate concentrations. Using mass transfer equations, the theoretical number of H+ at the microaqueous phase were calculated and expressed as hydrogen ion numbers to generate plots which indicated various thermodynamic processes operating at the microaqueous phase to maintain this concentration to a safe minimum.

A lipoxygenase-1 (LOX-1) inhibitor was isolated from the fermented broth of Aspergillus niger CFTRI 1105. It was purified, using column and preparative thin layer chromatography. 1H NMR and GC-MS examination revealed the structure of the inhibitor to be 2-(2'-methyl, 4'-hydroxyphenyl), 2-(4"hydroxyphenyl)-propane with a molecular weight of 242 and the molecular formula C,6H18O2. This bisphenol-derivative inhibitor shows 50% inhibition of soybean LOX-I at 0.98 mM concentration. The activity of this inhibitor was compared with commercial bisphenol A and its structural analogues, butylhydroxyanisole and butylhydroxytoluene in an attempt to understand the role of functional groups affecting lipoxygenase activity.

New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique.

Kinetics of lipase-catalyzed esterification of butyric acid and isoamyl alcohol have been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. No evidence of any significant diffusional limitations was detected that could affect the kinetics. The values of the apparent kinetic parameters were computed as: V(max)=11.72 micromol/min/mg; K(M, Acid)=0.00303 M; K(M, Alcohol)=0.00306 M; K(i, Acid)=1.05 M; and K(i, Alcohol)=6.55 M. This study indicates a competitive enzyme inhibition by butyric acid during lipase-catalyzed esterification reaction. Butyric acid, being a short-chain polar acid, concentrates in the microaqueous layer and causes a pH drop in the enzyme microenvironment leading to enzyme inactivation. Butyric acid binds to acyl-enzyme complex unproductively to yield a dead-end intermediate that can no longer give rise to an ester. High concentration of butyric acid gave rise to inactivation of the biocatalyst in addition to dead-end inhibition.

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.

The lactone 6-pentyl-alpha-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-alpha-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455 mg/l and 167 mg/l after 96 h and 48 h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters.

A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol-1. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol-1 at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h.

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 degrees C and glucose, maltose and maltodextrins at 70 degrees C as primary products, suggested significant applications for the enzyme in starch-processing industries.

A procedure to detect raw and gelatinized starch activities of glucoamylase on isoelectric focusing (IEF) gels by using 2, 3, 5-triphenyltetrazolium chloride is described. The reagent reacts with the reducing group of glucose released by glucoamylase from the substrate starch. Using the reaction, production of glucoamylase by Aspergillus niger was detected on 10% IEF gels within a pH range of 2.5-9.5. Since the method can detect raw and gelatinized starch activities of glucomylase associated with 1 microg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.

An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate.

In solid-state fermentation, the interaction of transport phenomena with biochemical reactions has a considerable effect on the productivity of the bioreactor. Previous work on solid-state fermentation in tray fermentors in our laboratory indicated that heat transfer resistance results in steep temperature gradients within the solid substrate bed, which in turn adversely affect the biochemical reaction and enzyme activity. This problem of heat accumulation during the course of fermentation has been alleviated to a considerable extent using a packed-column bioreactor with forced aeration in the present work. Experimental studies were conducted in a packed-column bioreactor utilizing wheat bran as substrate and the fungus Aspergillus niger CFTRI 1105 for the production of the enzyme amyloglucosidase. The enzyme activities were estimated and temperatures were recorded at different bed heights, for different air flow rates during the course of fermentation. The results indicated that the temperature gradients caused by heat transfer resistances were reduced considerably with corresponding increases in enzyme activity.

Persistence of chlorinated hydrocarbon insecticides in the environment is well documented. One early introduced insecticide, hexachlorocyclohexane (HCH), popularly called BHC, was used in large quantities all over the world until recently. In India, even today, technical grade HCH is being used extensively. Theoretically, HCH has eight possible stereoisomers of which four (alpha, beta, gamma, and delta) predominate in the technical product. These isomers significantly differ between themselves with respect to their persistence and toxicity toward insects, birds, mammals, and other nontarget organisms. The relative proportion of HCH isomers is, therefore, crucial from a toxicology standpoint. This problem assumes importance in light of reports that the HCH isomers undergo interconversion in soil, water, animals, plants, insects, etc. The persistence of HCH can be attributed in part to the interconversion of HCH isomers, which also restrict their solubility. In the present review, biotransformation of HCH isomers, both under aerobic and anaerobic conditions, and their degradation pathways have been described. In addition, emphasis is given to the interconversion of HCH isomers, including interconversion mechanisms, as this area has not received adequate coverage in earlier reviews on HCH.

In recent years due to acute shortage of calf-rennet, microbial rennets seem to be an effective alternative and are commercially produced. Mucor miehei was cultivated under the solid state fermentation conditions, and the moldy bran was extracted using a semicontinuous multiple contact forced percolation method. The treated extract was then filtered through 5% R16 clay which enabled easy and efficient removal of impurities such as lipase and protease without involving costly chemical treatments. The ethyl alcohol precipitated enzyme was dried and made into powder form having activity of 1.5 x 10(5)Soxhlet units/gm.

Mass transfer plays an important role in solid state fermentation (SSF) systems. Earlier work on SSF in tray bioreactors indicated that steep gaseous concentration gradients developed within the substrate bed, owing to mass transfer resistances, which may adversely affect the bioreactor performance. For all practical purposes these gradients have been eliminated using a packed bed column bioreactor with forced aeration. Gaseous concentrations (oxygen and carbon dioxide) and enzyme activities were measured at various bed heights for various air flow rates during the course of fermentation. The results indicated that concentration gradients were decreased effectively by increasing air flow rate. For example, the actual oxygen and carbon dioxide concentration gradients reduced from 0.07% (v/v) cm-1 and 0.023% (v/v) cm-1 to 0.007% (v/v) cm-1 and 0.0032% (v/v) cm-1 respectively when the air flow rate was increased from 5 dm3 min-1 to 25 dm3 min-1. This resulted in an overall improvement in the performance of the bioreactor in terms of enzyme production.