Probing surface protein patterning in biological systems using fluorescence nanoscopy

A complete understanding of biological substructures is often obscured by the diffraction limit of visible light in conventional fluorescence microscopy. Recently developed fluorescence nanoscopy techniques such as stochastic optical reconstruction microscopy (STORM) effectively break the diffraction limit (~250 nm) to enable imaging with an order of magnitude smaller than the spatial resolution. In this work, we applied fluorescence nanoscopy to study the patterning of proteins in biological surfaces at a high spatial resolution of 25 nm. We also developed new analytical methods to extract quantitative information on protein arrangement within biological substrates to elucidate key phenomenon such as signal amplification in bacteria during chemotaxis. In B. subtilis, we observed reorganization in arrays of McpB receptor proteins, key chemotactic receptors for asparagine sensing, upon exposure to saturating stimulant concentrations. More specifically, receptors formed large polar clusters in the absence of stimulant but shifted towards smaller and more dispersed lattices throughout the cell when stimulant was added. In a first-of-its-kind fluorescence nanoscopic study on B. subtilis, we measured cluster sizes and intra cluster density of these receptors. Our results support the existing theories on the role of cooperativity via receptor clustering in signal amplification to respond to wide range of external stimuli in bacteria. In second project, we studied the role of viral proteins on HIV-1 infectivity. In HIV-1, envelope proteins gp120 and gp41 are known to play a significant role in infectivity and their arrangement is indicative of internal viral structure at different stages of its life cycle. We ascertained the distribution of both the envelope proteins on surface of virions at a high resolution of ~25 nm using three-color fluorescence nanoscopy. Our work effectively paves the way for revealing new levels of organization of surface proteins at nanometer scale spatial resolution for a molecular view understanding of dynamics in biological systems

Subtilis spectacle: From microns to nanometers

The art shows diffraction limited (left) image transitioning towards a super-resolved image (right) of a chemotactic protein in bacteria called B.subtilis. The bacterial membrane (green) and DNA stain (blue) are also shown in the figure with localizations (red spots) indicating the protein of interest. The exquisite resolution of 20 nm is conferred by reconstruction of about 20,000 images obtained over time. (scale bar: 1000 nm) The ability to image cellular events using fluorescence microscopy is of paramount importance for understanding dynamic biological processes.However, optical diffraction limits the spatial resolution of conventional fluorescence microscopy to 250 nm, which precludes spatially resolved analysis of nanometer-scale cellular protein assemblies. Therefore, to elucidate a comprehensive molecular-level understanding of key events in bacterial motility, I am applying fluorescence nanoscopy-based imaging techniques to enable super-resolution imaging with an improved spatial resolution of 20-25 nm in our lab. I am currently investigating dynamic changes in spatial architectures of key chemotactic proteins in response to chemical stimulation. I capitalize on advances in synthetic click chemistry technology, optical microscopy, and digital image processing to enhance fundamental understanding on mechanics of bacterial motility, which has broad implications in applied industrial microbiology and provides a great precedent towards understanding cellular signaling

Clinical Characteristics and Management of the Hydatid Cyst of the Liver: A Study from a Tertiary Care Center in Nepal

A hydatid cyst of the liver is a significant yet neglected public health problem in Nepal. The present study was carried out to evaluate the demographic characteristics, clinical presentations, and management of the patients of the hydatid cyst of the liver in the setting of a developing country. It was a retrospective, descriptive analysis of 53 patients admitted in the department of surgery with the diagnosis of hydatid cyst of the liver based on clinical manifestations, imaging studies, or serology between 2016 and 2019. The median age of the patients was 36 years, with the age group of 25-45 years being the most commonly affected (23, 43.4%). 58.5% of the patients were female. Abdominal pain (49, 92.5%) and a palpable liver (17, 32.1%) were the most common complaint and physical finding in our study population, respectively. Abdominal ultrasonography and computed tomography scan were the major imaging studies used to establish a diagnosis. A unilocular and anechoic cystic lesion was the most frequent imaging finding. The right lobe of the liver harbored the cysts in the maximum number of patients. Surgery was the preferred modality of treatment (23, 43.4%), with pericystectomy being the most common form of surgical intervention. The hydatid cyst of the liver is a common cause of morbidity in Nepal. Clinical evaluation supplemented by imaging studies is required for diagnosis, and surgery remains the treatment of choice in most cases. To explain the epidemiological pattern of the disease, multicentric studies involving a larger sample of patients should be conducted

Clinical Characteristics and Management of the Hydatid Cyst of the Liver: A Study from a Tertiary Care Center in Nepal

A hydatid cyst of the liver is a significant yet neglected public health problem in Nepal. The present study was carried out to evaluate the demographic characteristics, clinical presentations, and management of the patients of the hydatid cyst of the liver in the setting of a developing country. It was a retrospective, descriptive analysis of 53 patients admitted in the department of surgery with the diagnosis of hydatid cyst of the liver based on clinical manifestations, imaging studies, or serology between 2016 and 2019. The median age of the patients was 36 years, with the age group of 25-45 years being the most commonly affected (23, 43.4%). 58.5% of the patients were female. Abdominal pain (49, 92.5%) and a palpable liver (17, 32.1%) were the most common complaint and physical finding in our study population, respectively. Abdominal ultrasonography and computed tomography scan were the major imaging studies used to establish a diagnosis. A unilocular and anechoic cystic lesion was the most frequent imaging finding. The right lobe of the liver harbored the cysts in the maximum number of patients. Surgery was the preferred modality of treatment (23, 43.4%), with pericystectomy being the most common form of surgical intervention. The hydatid cyst of the liver is a common cause of morbidity in Nepal. Clinical evaluation supplemented by imaging studies is required for diagnosis, and surgery remains the treatment of choice in most cases. To explain the epidemiological pattern of the disease, multicentric studies involving a larger sample of patients should be conducted.</jats:p