Novel approaches for prebiotic detection and control of microbial communities

Since the turn of the millennium, we have witnessed the emergence of synthetic biology as a field which set the stage for transforming biotechnology and medicine. The tools developed have enabled us to apply general engineering approaches to manipulate biological systems towards achieving desired functions. In this work, we harness the programmability of microbes to understand the role of prebiotic oligosaccharides. Prebiotics are important glycans that shape the human gut microbiota from as early as, shortly after birth. Human milk oligosaccharides (HMOs), found in breast milk, are naturally occurring chemicals that selectively promote growth or activity in a microbial community. Given the benefits of prebiotics, especially HMOs, there has been a lot of interest in replicating their function by synthesizing them. Numerous approaches have been developed for their large-scale synthesis, however, the structural analysis of glycans is challenging and is under steadily increasing demand. The range of analytical techniques currently accessible to analyze glycans is limited by a lack of suitable high-throughput techniques, relying mostly on chromatographic methods. Biological systems have enormous potential to solve many engineering problems, and synthetic biology provides an attractive approach to harness this potential. In this work, I first describe two platforms for the development of high-throughput, linkage-specific screening of glycans. The first is based on a genetically engineered whole-cell biosensor, which produces a response to lactose. I created a library of specific glycosidases that can trim complex HMOs down to lactose and trigger a response. The second platform is based on an enzymatic, paper-based assay for determination of the type of fucosylation and sialylation in glycans. The paper device was developed with immobilized enzymes that was coupled to a colorimetric, redox assay for rapid screening. This part of the work provides new techniques to enable a streamlined synthesis process for HMOs with a significant reduction in analysis time of new producer strains. The second platform paves the way for development of a glyco-barcode assay for rapid analysis of key glycosylation patterns not only in biosynthesis platforms but also for diagnosis or monitoring disease states in different biomarkers. The other focus of my thesis is directed towards manipulating engineered microbial communities. Targeted biochemical modulation of microbiota can utilize prebiotics that are designed to modulate microbial function or growth. Here, I demonstrated engineered bacterial strains that, owing to selective utilization of HMOs, enabled growth-based selection in mixed cultures. The dynamic regulation of growth and protein production in mixed culture models is also demonstrated. We also illustrate chemical synthesis of bio-inspired novel inducer molecules for orthogonal control of protein expression The first chapter of the dissertation introduces the concept of prebiotics and highlights some key advantages and their mechanisms of action. Chapter two covers genetically encoded whole cell biosensors that enable high-throughput, linkage-specific detection of HMOs and enable dynamic regulation of growth and protein expression in mixed populations. The third and fourth chapters look at paper-based sensing, keeping rapid, simple, low-cost real-time detection, in mind. Chapters four and five describe approaches for microbiome manipulations, a shift towards understanding and engineering community-level interactions in mixed populations. Finally, chapter six introduces chemically synthesized novel inducer molecules that were inspired by biological mimics. With this work, the goal was to contribute towards expanding the glycobiology toolbox and help elucidate the staggering complexity of glycans. The promises of the ability to engineer microbial populations are immense in that we are living in symbiosis with bacteria, and their interactions play roles that heavily impact human health. Taken together, these projects demonstrate a variety of approaches for glycan analysis and utilization, taking inspiration from the denizens of the human gut microbiome and suggests multiple avenues forward towards clinical and health impact

Diffusion-weighted magnetic resonance imaging may be useful in differentiating fungal abscess from malignant intracranial lesion: Case report

Background: Diffusion-weighted magnetic resonance has a well-defined role in differentiating between important intracranial lesions. Sometimes, the surgeon is faced with a dilemma of how to diagnose an infectious versus malignant lesion.Case Description: A 28-year-old male presented to the neurosurgery clinic with complaints of headache and left-sided weakness for 2 weeks. Neurological examination was intact. Magnetic resonance imaging (MRI) scan showed a large infiltrating heterogeneous mass involving the right parietal lobe. On further reviewing, there was homogenous diffusion restriction in the center of lesion. In addition, its aggressive behavior confirmed it to be a fungal abscess.Conclusions: Correctly identifying an infectious versus tumor etiology is important. Research has been carried out to employ diffusion-weighted imaging (DWI) in differentiating the variable radiological findings. The role of DWI in diagnosing bacterial abscess is more commonly seen in comparison to fungal abscess. DWI has a high diagnostic potential, but more works need to be done

Flipped classroom instructional approach in undergraduate medical education

Objective: In this study we implemented the “flipped classroom” model to enhance active learning in medical students taking neurosciences module at Aga Khan University, Karachi. Methods: Ninety eight undergraduate medical students participated in this study. The study was conducted from January till March 2017. Study material was provided to students in form of video lecture and reading material for the non-face to face sitting, while face to face time was spent on activities such as case solving, group discussions, and quizzes to consolidate learning under the supervision of faculty. To ensure deeper learning, we used pre- and post-class quizzes, work sheets and blog posts for each session. Student feedback was recorded via a likert scale survey. Results: Eighty four percent students gave positive responses towards utility of flipped classroom in terms of being highly interactive, thought provoking and activity lead learning. Seventy five percent of the class completed the pre-session preparation. Students reported that their queries and misconceptions were cleared in a much better way in the face-to-face session as compared to the traditional setting (4.09 ±1.04). Conclusion: Flipped classroom(FCR) teaching and learning pedagogy is an effective way of enhancing student engagement and active learning. Thus, this pedagogy can be used as an effective tool in medical schools

Diffusion tensor imaging for ruptured cerebral arteriovenous malformation

Non-ruptured arteriovenous malformations (AVMs) rarely cause tract disruption. Few studies have described how ruptured AVMs influence white matter (WM) tract morphology. We reviewed consecutive AVM cases treated at a tertiary care hospital where diffusion tensor imaging (DTI) tractography was obtained preoperatively. DTI was performed using the Synaptive Plan (Synaptive Medical Inc., Toronto, Canada). Quality control was performed by clinical application specialist. Perinidal fractional anisotropy (FA) value of corticospinal tracts (CST) was obtained. A reference FA value was obtained from the corresponding area on the contralateral side. Images were evaluated by a consultant neuroradiologist. Radiological findings were correlated with clinical findings. White matter morphology was described by a consultant neuroradiologist. All three cases included in the study had a history of haemorrhage in the past. Two patients had disruption of CST and presented with a significant neurological deficit. In one patient FA value of CST around the nidus was comparable to the contralateral side and did not show any neurological deficit. DTI integrated neuronavigation was used to plan the trajectory and complete resection of the AVM with excellent postoperative recovery

Video microscope robotic arm-assisted, neuronavigation-guided glioma resection and regional sampling

High-grade gliomas possess internal pathological heterogeneity. Selective sampling of different tumor regions can help in the study of this heterogeneity. In this report, we have described the use of a novel navigation and optical system for the selective regional sampling of a high-grade glioma lesion. A 45-year-old gentleman presented to us with complaints of intermittent frontal headaches for past eight months. On examination, he had subtle pyramidal weakness in left upper and lower extremities. Magnetic resonance imaging (MRI) showed a large contrast-enhancing, space-occupying lesion in the right frontal lobe causing perilesional edema and midline shift. We marked four different regions on the preoperative MRI using apparent diffusion coefficient (ADC) mapping and contrast enhancement pattern in four different combinations using presurgical planning software (BrightMatter™ Plan) (Synaptive Medical, Inc., Toronto, Canada). These pre-identified areas were exported into BrightMatter™ Servo (Synaptive Medical, Toronto, Canada), an integrated robotic video microscope with a neuronavigation system where these areas were selectively sampled and sent for analysis. The BrightMatter™ Servo not only helped us to the target areas but also helped to identify a safe trajectory, respecting white matter tracts. Histopathology showed a neoplastic lesion composed of mononuclear round cells with the perinuclear halo in a fibrillary stroma with admixed mini-gemistocytes consistent with the diagnosis of a Grade 3 anaplastic astrocytoma. A selective regional sampling of the gliomas can be reliably performed using BrightMatter™ technologies to study the pathological heterogeneity of these lesions

Current management of glioma in Pakistan

To date, information on the management of specific neurosurgical tumors, such as glioma, in Pakistan remains scattered and scarce. Our review synthesizes the predicaments of glioma management routinely presented to the neurosurgery, medical oncology, radiation oncology, and radiology departments in Pakistan. Expert opinions were integrated from each of the relevant fields in the form of personal citations. The data presented in our review were collected from various PubMed and non-PubMed indexed articles, coupled with various health reports from the Government of Pakistan along with the World Health Organization. Through these data, it was postulated that the utilization of innovative and instrumental technologies is a constant struggle for neurosurgeons in Pakistan, considering the cost-effectiveness. Hence, this results in significant limitations for surgeons to provide the best outcome for their patients. As most Pakistanis (74%) pay out of pocket, measuring cost‑effectiveness is extremely crucial. It was found that significant differences in intra‑operative and postoperative care existed among various centers. Public sector institutions fared much worse. The role of diagnostics in glioma surgery is severely limited across centers in Pakistan and as such, research and training need to be addressed promptly. In order to achieve success in glioma management, the data in our article demonstrate various facets of health care that need to be addressed simultaneously and swiftly. Surgical access needs to be improved; only then, optimal management of glioma can be accomplished in Pakistan

Novel approaches for prebiotic detection and control of microbial communities

Since the turn of the millennium, we have witnessed the emergence of synthetic biology as a field which set the stage for transforming biotechnology and medicine. The tools developed have enabled us to apply general engineering approaches to manipulate biological systems towards achieving desired functions. In this work, we harness the programmability of microbes to understand the role of prebiotic oligosaccharides. Prebiotics are important glycans that shape the human gut microbiota from as early as, shortly after birth. Human milk oligosaccharides (HMOs), found in breast milk, are naturally occurring chemicals that selectively promote growth or activity in a microbial community. Given the benefits of prebiotics, especially HMOs, there has been a lot of interest in replicating their function by synthesizing them. Numerous approaches have been developed for their large-scale synthesis, however, the structural analysis of glycans is challenging and is under steadily increasing demand. The range of analytical techniques currently accessible to analyze glycans is limited by a lack of suitable high-throughput techniques, relying mostly on chromatographic methods. Biological systems have enormous potential to solve many engineering problems, and synthetic biology provides an attractive approach to harness this potential. In this work, I first describe two platforms for the development of high-throughput, linkage-specific screening of glycans. The first is based on a genetically engineered whole-cell biosensor, which produces a response to lactose. I created a library of specific glycosidases that can trim complex HMOs down to lactose and trigger a response. The second platform is based on an enzymatic, paper-based assay for determination of the type of fucosylation and sialylation in glycans. The paper device was developed with immobilized enzymes that was coupled to a colorimetric, redox assay for rapid screening. This part of the work provides new techniques to enable a streamlined synthesis process for HMOs with a significant reduction in analysis time of new producer strains. The second platform paves the way for development of a glyco-barcode assay for rapid analysis of key glycosylation patterns not only in biosynthesis platforms but also for diagnosis or monitoring disease states in different biomarkers. The other focus of my thesis is directed towards manipulating engineered microbial communities. Targeted biochemical modulation of microbiota can utilize prebiotics that are designed to modulate microbial function or growth. Here, I demonstrated engineered bacterial strains that, owing to selective utilization of HMOs, enabled growth-based selection in mixed cultures. The dynamic regulation of growth and protein production in mixed culture models is also demonstrated. We also illustrate chemical synthesis of bio-inspired novel inducer molecules for orthogonal control of protein expression The first chapter of the dissertation introduces the concept of prebiotics and highlights some key advantages and their mechanisms of action. Chapter two covers genetically encoded whole cell biosensors that enable high-throughput, linkage-specific detection of HMOs and enable dynamic regulation of growth and protein expression in mixed populations. The third and fourth chapters look at paper-based sensing, keeping rapid, simple, low-cost real-time detection, in mind. Chapters four and five describe approaches for microbiome manipulations, a shift towards understanding and engineering community-level interactions in mixed populations. Finally, chapter six introduces chemically synthesized novel inducer molecules that were inspired by biological mimics. With this work, the goal was to contribute towards expanding the glycobiology toolbox and help elucidate the staggering complexity of glycans. The promises of the ability to engineer microbial populations are immense in that we are living in symbiosis with bacteria, and their interactions play roles that heavily impact human health. Taken together, these projects demonstrate a variety of approaches for glycan analysis and utilization, taking inspiration from the denizens of the human gut microbiome and suggests multiple avenues forward towards clinical and health impact.</p

Alpha terpineol preconditioning enhances regenerative potential of mesenchymal stem cells in full thickness acid burn wounds

Regeneration of full thickness burn wounds is a significant clinical challenge. Direct stem cell transplantation at the wound site has a promising effect on wound regeneration. However, stem cell survival within the harsh wound environment is critically compromised. In this regard, preconditioning of stem cells with cytoprotective compounds can improve the efficiency of transplanted cells. This study evaluated the possible effect of alpha terpineol (αT) preconditioned mesenchymal stem cells (αT-MSCs) in full thickness acid burn wound. An optimized concentration of 10 μM αT was used for MSC preconditioning, followed by scratch assay analysis. A novel rat model of full thickness acid burn wound was developed and characterized via macroscopic and histological examinations. Treatment (normal and αT-MSCs) was given after 48 h of burn wound induction, and the healing pattern was examined till day 40. Skin tissues were harvested at the early (day 10) and late (day 40) wound healing phases and examined by histological grading, neovascularization, and gene expression profiling of healing mediators. In scratch assay, αT-MSCs exhibited enhanced cell migration and wound closure (scratch gap) compared to normal MSCs. In vivo findings revealed enhanced regeneration in the wound treated with αT-MSCs compared to normal MSCs and untreated control. Histology revealed enhanced collagen deposition with regenerated skin layers in normal MSC- and αT-MSC treated groups compared to the untreated control. These findings were correlated with enhanced expression of α-SMA as shown by immunohistochemistry. Additionally, αT-MSC group showed reduced inflammation and oxidative stress, and enhanced regeneration, as witnessed by a decrease in IL-1β, IL-6, TNF-α, and Bax and an increase in BCL-2, PRDX-4, GPX-7, SOD-1, VEGF, EGF, FGF, MMP-9, PDGF, and TGF-β gene expression levels at early and late phases, respectively. Overall findings demonstrated that αT exerts its therapeutic effect by mitigating excessive inflammation and oxidative stress while concurrently enhancing neovascularization. Thus, this study offers new perspectives on managing full thickness acid burn wounds in future clinical settings

Hypothalamic cavernoma (A unique case observation)

Cavernomas are vascular malformations that occur commonly in cerebrum, cerebellum, and brainstem, being rare in occurrence within the hypothalamus having only 29 cases known in the literature. They may be clinically asymptomatic or present with symptoms of headache, seizures, hemorrhage, or focal neurologic deficits. The gold standard for diagnosing a cavernoma is magnetic resonance imaging (MRI). The management strategies for hypothalamic cavernoma are expectant, medical, and surgical, along with laser ablation and radiosurgery. Our case is of a 17-years old female who presented with headache and reduced vision secondary to hypothalamic cavernoma on diagnostic imaging. She is on expectant management till date, without worsening of her symptoms. As per the literature, there has been no data on conservatively-managed hypothalamic hamartomas