Efficient assembly of a synthetic attenuated SARS-CoV-2 genome in Saccharomyces cerevisiae using multi-copy yeast vectors.

Saccharomyces cerevisiae has been demonstrated to be an excellent platform for the multi-fragment assembly of large DNA constructs through its powerful homologous recombination ability. These assemblies have invariably used the stable centromeric single copy vectors. However, many applications of these assembled genomes would benefit from assembly in a higher copy number vector for improved downstream extraction of intact genomes from the yeast. A review of the literature revealed that large multi-fragment assemblies did not appear to have been attempted in multicopy vectors. Therefore, we devised a toolkit that would enable one to seamlessly transition with the same assembling fragments between a single copy and a multicopy vector. We evaluated the assembly of a 28 kb attenuated SARSCoV- 2 genome (lacking the N gene) from 10 fragments in both single copy and multicopy vector systems. Our results reveal that assembly was comparably efficient in the two vector systems. The findings should add to the synthetic biology toolkit of S. cerevisiae and should enable researchers to utilize any of these vector systems depending on their downstream applications.

Novel wire-free techniques for localization of impalpable breast lesions-A review of current options.

Localization methods for breast lesions including cancers have changed and advanced since their inception. Currently, the most widely used technique in the United Kingdom is the image-guided hook wire localizer developed in the 1970s. It remains as the gold standard for localization of impalpable breast tumors. Besides its advantages, there are some disadvantages associated with this technique. In recent years, novel wire-free techniques (eg, Magseed® , SCOUT® , and LOCalizer™) have been developed to not only localize impalpable breast lesions but also negate the disadvantages of wire localization. This article reviews the variety of techniques from their origins to the most recent advancements that are used to localize breast lesions. The future is heading toward non-wire technology and wire localization may then be reserved for special cases.

Site-specific PEGylation of micro-plasmin for improved thrombolytic therapy through engineering enhanced resistance against serpin mediated inhibition.

The relatively rapid inhibition of microplasmin by α2-AP leads to short functional half-life of the molecule in vivo, causing inefficient clot dissolution, even after site-specific, local catheter-based delivery. Here, we describe a PEGylation approach for improving the therapeutic potential via improving the survival of microplasmin in presence of its cognate inhibitor, α2-AP, wherein a series of strategically designed cysteine analogs of micro-plasminogen were prepared and expressed in E. coli, and further modified by covalent grafting in vitro with PEG groups of different molecular sizes so as to select single or double PEG chains that increase the molecular weight and hydrodynamic radii of the conjugates, but with a minimal discernible effect on intrinsic plasmin activity and structural framework, as explored by amidolytic activity and CD-spectroscopy, respectively. Interestingly, some of the purified PEG-coupled proteins after conversion to their corresponding proteolytically active forms were found to exhibit significantly reduced inhibition rates (up to 2-fold) by α2-AP relative to that observed with wild-type microplasmin. These results indicate an interesting, and not often observed, effect of PEG groups through reduced/altered dynamics between protease and inhibitor, likely through a steric hindrance mechanism. Thus, the present study successfully identifies single- and double-site PEGylated muteins of microplasmin with significantly enhanced functional half-life through enhanced resistance to inactivation by its in vivo plasma inhibitor. Such an increased survival of bioactivity in situ, holds unmistakable potential for therapeutic exploitation, especially in ischemic strokes where a direct, catheter-based deposition within the cranium has been shown to be promising, but is currently limited by the very short in vivo bioactive half-life of the fibrin dissolving agent/s.

Biomarkers of Cardiac Health and Disease.

Cardiovascular diseases (CVDs) are globally associated with high rates of morbidity and mortality. CVDs are diagnosed based on clinical assessment, physical examination, disease history, electrocardiogram, and chest X-ray. However, these parameters have limited sensitivity and specificity. Furthermore, CVDs include a range of conditions and are characterized by a wide variation in symptoms. Some patients with CVDs can also be asymptomatic. Hence, biomarkers are required for CVD confirmatory diagnoses. Although various biomarkers are suggestive of different CVDs, most of them are not used clinically in CVD diagnosis. This is because a single molecule is not completely suitable for diagnosis, especially at an early stage, or these markers have not been exploited beyond study stages. Because CVDs globally affect millions of individuals, a method for confirmatory detection at onset is eagerly required for effective clinical management of patients. This review focuses on potential biomolecules that can indicate the presence of different CVDs and cardiac health status.

Optical Coherence Tomography and Subclinical Optical Neuritis in Longitudinally Extensive Transverse Myelitis.

OBJECTIVE: The aim is to compare the retinal nerve fiber layer (RNFL) thickness of longitudinally extensive transverse myelitis (LETM) eyes without previous optic neuritis with that of healthy control subjects. METHODS: Over 20 LETM eyes and 20 normal control eyes were included in the study and subjected to optical coherence tomography to evaluate and compare the RNFL thickness. RESULT: Significant RNFL thinning was observed at 8 o'clock position in LETM eyes as compared to the control eyes (P = 0.038). No significant differences were seen in other RNFL measurements. CONCLUSION: Even in the absence of previous optic neuritis LETM can lead to subclinical axonal damage leading to focal RNFL thinning.

Non-healing gastro-duodenal ulcer: A rare presentation of primary abdominal tuberculosis.

INTRODUCTION: We present a case of primary gastrointestinal tuberculosis that has culminated in ulcer formation, in the absence of pulmonary involvement in an immunocompetent patient. PRESENTATION OF CASE: A 28-year-old Asian male presented to casualty with a 1-week history of epigastric cramping abdominal pain and several episodes of non-bilious vomiting. The patient deteriorated clinically, becoming more cachectic and given his unexplained weight loss, an oesophageal-gastro-duodenal endoscopic imaging confirmed a duodenal ulcer. The biopsy of the non-healing ulcer was the hallmark of the disease, revealing evidence of granulomatous inflammation consistent with tuberculosis bacilli. DISCUSSION: Gastrointestinal tuberculosis with ulceration is rare with respect to the oesophagus, stomach and duodenum. This case proves to be unique, as our patient had experienced primary isolated gastric tuberculosis in the absence of pulmonary tuberculosis in a healthy individual. Immunohistochemical staining, histopathology and radiological investigations have demonstrated their importance in confirming abdominal tuberculosis and the extent of bowel involvement. CONCLUSION: This case has illustrated the difficulties associated with a prompt diagnosis of an unusual case of primary duodenal tuberculosis from chronic peptic ulcer disease in an immunocompetent patient.

Strongyloides stercoralis hyperinfection presenting as subacute small bowel obstruction following immunosuppressive chemotherapy for multiple myeloma.

We report the case of a 59-year-old Afro-Caribbean woman who presented with symptoms of anorexia, lethargy, abdominal distension and vomiting on the background of newly diagnosed multiple myeloma, treated with one cycle of cyclophosphamide-thalidomide-dexamethasone chemotherapy 20 days previously. A diagnosis of subacute bowel obstruction was made; however, the aetiology of the obstruction remained elusive. Common electrolyte abnormalities were excluded and a midline laparotomy revealed minimal intra-abdominal adhesions. Histological examination of a small bowel mesentery biopsy showed inflammatory cell infiltrate composed of lymphocytes, eosinophils and occasional plasma cells with a foreign body giant cell reaction suggestive of worm infection. A postoperative stool sample revealed heavy infestation with the rhabditiform larvae of Strongyloides stercoralis. The patient recovered following ivermectin treatment. In the absence of other causality, we attribute the subacute bowel obstruction to S stercoralis hyperinfection, triggered by immunosuppression secondary to chemotherapy and multiple myeloma.

Acute abdominal pain and radiological pneumoperitoneum - always an indication for laparotomy?

Pneumoperitoneum in the presence of acute abdominal pain is well recognised as an indication for laparotomy. We present a case of acute abdominal pain in the presence of an incidental pneumoperitoneum secondary to the rupture of pneumatosis intestinalis. We will discuss the importance of clinical context in the diagnosis and management of pneumoperitoneum and pneumatosis intestinalis.

Retroperitoneal unicentric Castleman's disease with multiple lymph node involvement.

A 78-year-old woman with B-symptoms was referred for a left adrenal incidentaloma of 5 cm. Imaging revealed features compatible with adrenal cancer. The authors excluded excess production of catecholamines or adrenal steroids. The tumour was removed by en bloc radical left retroperitonectomy with adrenalectomy, nephrectomy, interaortocaval lymphadenectomy and splenectomy. Histology demonstrated periadrenal hyaline vascular Castleman's disease with local infiltration and 14 positive lymph nodes. The lymphoid infiltrate spilled into the adjacent renal cortex. HHV8 was negative. The Ki67 proliferative index was 30-40% in germinal centres. There was no syn- or metachronous disease on extended imaging including fluorodeoxyglucose positron emission tomography-CT and narrow follow-up at 3 years. This is a rare case of unicentric hyaline vascular Castleman's disease with documented locoregional lymph node involvement. The case exemplifies the transition from unifocal unicentric disease into disseminated disease with involvement of multiple lymph node stations (multicentric disease). The authors demonstrate surgical cure by oncological resection.

A forgotten migrated intrauterine contraceptive device is not always innocent: a case report.

The incidence of transuterine perforation and migration of intrauterine contraceptive devices (IUCDs) into the abdominal cavity has been estimated at less than 0.1%. It has been suggested that intraperitoneal IUCD have low morbidity and may be left in situ. We report the first case of closed loop small bowel obstruction due to migration of a "Saf-T-Coil" IUCD into the abdominal cavity, where it became embedded in the omentum and ultimately, 31 years after deployment, coiled both arms around a loop of ileum. This late complication underlines the dangers of intra-abdominal foreign bodies, even when chemically and biologically inert.

Every wheeze does not merit a puffer! Case of an overnight cure of chronic asthma.

The peril of incorrect diagnostic labelling is highlighted by this case of acute respiratory distress caused by a retrosternal recurrent goitre. An initial clinical diagnosis which cannot be fully validated on investigation with unexpected or poor response to treatment should prompt consideration and investigation for an alternative explanation.

Lymph node involvement and surgical approach in parathyroid cancer.

BACKGROUND: The best surgical approach to parathyroid cancer is disputed. Recommendations vary and are built on incoherent evidence. High rates of recurrence and death require an in-depth review of underlying findings. METHODS: This retrospective study includes 11 patients with parathyroid cancer who underwent surgery with central and/or lateral neck dissection by a single surgeon between 2005 and 2010. The diagnosis was based on histopathological criteria in all patients. Patterns of lymph node and soft tissue involvement of these and formerly reported patients were analysed based on full-text review of all published cases of parathyroid cancer. RESULTS: In this series only 1 of 11 patients (9.1%) manifested lymph node metastasis. In the literature, lymph node metastases have been reported in only 6.5% of 972 published patients, or in 32.1% of the 196 in whom lymph node involvement was assessed by the authors. They were, with few exceptions, localised in the central compartment. Recurrence in soft tissue is more frequent than in locoregional lymph nodes. CONCLUSION: Oncological en bloc clearance of the central compartment with meticulous removal of all possibly involved soft tissues, including a systematic central lymph node resection, may improve outcomes and should be included in the routine approach to the suspicious parathyroid lesion. There is no need for a prophylactic lateral neck dissection.

Management of an incidentally found large adrenal myelolipoma: a case report.

Adrenal myelolipoma is a rare benign neoplasm composed of mature adipose and hematopoietic tissue. Most lesions are small, unilateral and asymptomatic, discovered incidentally at autopsy or on imaging studies performed for other reasons. We would like to present a case report of this rare tumour. Cross-sectional imaging is helpful in making a pre-operative diagnosis. The size of the lesion should be a criterion for surgical intervention.

Complications of spilled gallstones following laparoscopic cholecystectomy: a case report and literature overview.

INTRODUCTION: Gallbladder perforation is common and occurs in 6 to 40% of laparoscopic cholecystectomy procedures. In up to a third of these cases, stones are not retrieved and complications can arise many years post-operatively. Diagnosis can be difficult and patients may present to many specialties within medicine and surgery. We seek to present our case and review the literature on prevention and management of "lost" stones. CASE PRESENTATION: Our patient is a 77-year-old woman who presented to the urology clinic with a loin abscess that developed five years after laparoscopic cholecystectomy. Radiological studies showed retained abdominal gallstones and an associated abscess formation. These were drained under ultrasound guidance on several occasions and the patient now suffers from chronic sinusitis. Due to her age and comorbidities, she has declined definitive surgical intervention to remove the stones. CONCLUSION: Gallbladder perforation during laparoscopic cholecystectomy is a reasonably common problem and may result in spilled and lost gallstones. Though uncommon, these stones may lead to early or late complications, which can be a diagnostic challenge and cause significant morbidity to the patient. Clear documentation and patient awareness of lost gallstones is of utmost importance, as this may enable prompt recognition and treatment of any complications.

The nasal septum: an osteometric study of 16 cadaver specimens.

Studies of the shape, dimensions, and other morphologic characteristics of the nasal septum are scant in the literature. We conducted a study of 16 male cadavers to measure six osseous components of the nasal septum and to calculate the proportional contribution that each made to the total septal bone surface area. We found a wide range of inter-specimen variations in both categories. It is our hope that a better understanding of nasoseptal anatomy will improve surgical technique.